CN1915433A - Relativity between AnnexinA3 and drug resistance of platinum type chemical curing medication for cancer - Google Patents

Abstract

The present invention relates to the correlation between annexin A3 and the drug resistance of platinum-based chemotherapeutic drugs in cancer, to a method for improving/decreasing the drug resistance of platinum-based chemotherapeutic drugs in cancer by changing the expression of annexin A3 in cells, and to methods for detecting and Methods for detecting or diagnosing the resistance of cancer to platinum-based chemotherapy drugs, and agents that can be used in these methods, pharmaceutical compositions comprising the agents, uses of the agents for preparing medicines, and the agents screening method. Furthermore, the present invention also relates to methods for treating cancer patients. In the present invention, the cancer is a cancer that can be treated with platinum-based chemotherapy drugs, more preferably the cancer is ovarian cancer.

Description

Correlation between Annexin A3 and resistance to platinum-based chemotherapy drugs in cancer

Technical field

The present invention generally relates to the field of chemotherapeutic drug resistance, and more specifically the present invention relates to the correlation between annexin A3 and the resistance of platinum-based chemotherapeutic drugs in cancer, and relates to increasing/decreasing the platinum in cancer by changing the expression of annexin A3 in cells A method for chemotherapeutic drug resistance, and a medicament that can be used in the method to change the expression of annexin A3 in cells, a pharmaceutical composition comprising the medicament, and the use of the medicament for preparing a drug that changes drug resistance of cancer, and A screening method for the medicament. In addition, the present invention also relates to detection or diagnosis methods, medicaments, pharmaceutical compositions and kits for detection and/or prognosis of cancer resistance to platinum-based chemotherapy drugs. The invention also relates to methods for treating cancer patients. In the present invention, the cancer is a cancer that can be treated with platinum-based chemotherapy drugs, more preferably the cancer is ovarian cancer.

Background technique

Ovarian cancer is the first fatal disease of gynecological tumors. The 5-year survival rate of advanced patients is always hovering at 15%-20%. The resistance of tumor cells to chemotherapy drugs is one of the main reasons for this situation. Platinum combines with DNA to form a cross bond, thereby destroying the function of DNA and can no longer replicate. At high concentrations, it can also inhibit the synthesis of RNA and protein. It is currently the most commonly used first-line chemotherapy drug for ovarian cancer, and it is also used for the treatment of other solid tumors including breast cancer. Commonly used drugs for cancer, lung cancer, testicular tumor, head and neck cancer, osteosarcoma, melanoma, esophageal cancer, etc. In the platinum-based treatment of these solid tumors, drug resistance has emerged, that is, the tumor is well controlled in the early stage of treatment, but relapse occurs in the later stage of treatment or there is no response in the early stage of treatment. Clinically, ovarian cancer has a clear understanding and definition of platinum drug resistance, however, the elucidation of the mechanism of cisplatin drug resistance is far from enough. Although the current understanding of the mechanism of tumor drug resistance from the gene level has been quite in-depth, such as MDR-1, LRP-1, MRP-1, GST-pi, etc. have become well-known drug resistance-related genes. However, the results of most studies have shown that the expression of multiple drug resistance-related genes in ovarian cancer cannot predict the drug resistance and prognosis of tumors well, and the inconsistency between the abundance of mRNA and its corresponding protein level may be one of the main reasons. DNA is the carrier of genetic information, and protein is the executor of functions. Therefore, high-throughput technology at the protein level is needed to further explore the mechanism of drug resistance, and proteomics technology provides the possibility for the functional study of drug resistance genes.

Proteomics technology is an emerging technology developed in recent years and has been widely used in various fields of basic research. Generally speaking, proteomics research mainly includes the following aspects: 1. Expression proteomics; 2. Functional protein omics; 3. structural proteomics. At present, the application of proteomic technology in ovarian tumors mainly focuses on the early diagnosis of malignant tumors and the screening of tumor markers, but so far there is no research on ovarian cancer drug resistance markers. Starting from clinical tissues to directly study the mechanism of platinum drug resistance, there are many difficulties such as heterogeneity, complex influencing factors, limited experimental technology level, and easy loss of positive results. Therefore, taking the cell line as the research object and gradually extending it to the clinic provides a scientific research idea.

The Annexin family (I～XIII) is a group of Ca ²⁺ -dependent phospholipid-binding proteins with non-EF chiral Ca ²⁺ binding sites, which play a certain role in regulating cell growth and cell signal transduction pathways. Annexin A3 is the only member with enzymatic activity in the annexin family, which has the function of inhibiting phospholipase A2 and anticoagulation. It can also cut off inositol1, 2-cyclic phosphate to form inositol 1-phosphate, and play a role in controlling cell proliferation [Ross TS , et al. J Biol Chem. 1991, 266(14): 9086-9092.]. Although it widely exists in various tissues and organs of animals and plants, annexin A3 is predominantly expressed in neutrophils in the final stage of differentiation. In unstimulated neutrophils, it is distributed in the cytoplasm, and its content is as high as 1% of the total cytoplasmic protein [Ernst JD, et al. J Clin Invest, 1990, 85(4): 1065-1071.]. Under a certain Ca ²⁺ concentration, annexin A3 can bind to the phosphatidylserine of the vesicle, mediate the fusion of phagosome and lysosome and the disappearance of granules and other membrane-to-membrane effects. In addition, it is able to promote the accumulation of specific granules isolated from human neutrophils [Le Cabec V, et al. Biochem J, 1994, 303(Pt 2): 481-487.]. Recently, studies have shown that annexin A3 is a new pro-angiogenic factor, which can lead to the increase of VEGF by activating HIF-1, providing a new target for anti-tumor growth and angiogenesis [Park JE, et al.Biochem Biophys Res Commun. 2005, 337(4): 1283-1287.]. So far, there is no research report on the correlation between annexin A3 and tumor chemotherapy resistance.

In order to overcome the shortcomings that the expression of drug-resistance-related genes in cancer cannot well predict the drug resistance and prognosis of tumors, and the separation of basic research and clinical treatment, the inventors of the present application identified the relationship between protein annexin A3 and cancer ( Especially ovarian cancer) is related to the resistance of platinum-based chemotherapy drugs, and has been further verified clinically, so that it is possible to predict the chemotherapy resistance of clinical cancer patients by detecting the expression of annexin A3, detect drug resistance early, and change the treatment plan , improve the 5-year survival rate, and make it possible to change the therapeutic effect of platinum-based drugs on such patients by changing the expression of annexin A3 in the tumors of drug-resistant clinical cancer patients. In addition, the construction of eukaryotic expression plasmids for positive and antisense annexin A3 and eukaryotic expression plasmids expressing annexin A3 fusion protein proposed in the present invention provides a favorable tool for studying the function of annexin A3. Furthermore, the present invention also provides a method for inducing ovarian cancer cisplatin-resistant cell line SKOV3/CDDP-P, simulating the dose and mode of clinical treatment of ovarian cancer, and providing a good cell model for further research on clinical drug resistance.

Contents of the invention

Therefore, one aspect of the present invention relates to a method for regulating the drug resistance of platinum-based chemotherapy drugs in cancer, comprising changing the expression of annexin A3 in cancer cells, wherein preferably, the cancer is ovarian cancer, and the platinum-based chemotherapy drugs are cis platinum or carboplatin. The method of the present invention can be used to regulate the drug resistance of cancer cells in vitro or can be used to regulate the drug resistance of cancer patients in vivo.

In one embodiment of the method of the present invention, the tolerance of cancer cells to platinum-based chemotherapy drugs is improved by increasing the expression of annexin A3 in cancer cells, preferably, the expression of annexin A3 in cancer cells is increased. The expression of annexin A3, preferably wherein the content of annexin A3 in the cells is increased by using a plasmid expressing a positive-sense annexin A3 gene.

Therefore, the present invention also relates to polynucleotides encoding annexin A3 protein, DNA constructs and expression vectors comprising the polynucleotides, and their use in the preparation of drugs for improving cancer resistance to platinum-based chemotherapy drugs. In the present invention, the annexin A3 protein includes not only the natural annexin A3 protein but also artificially synthesized (for example, by genetic engineering) annexin A3 proteins, such as fusion proteins containing annexin A3.

In another embodiment of the method of the present invention, the resistance of cancer to platinum chemotherapy drugs is reduced by reducing and/or inhibiting the expression of annexin A3 in cancer cells, preferably, by expressing the expression of annexin A3 in cancer cells Antisense nucleic acid and/or the ribozyme specific to annexin A3 reduce and/or inhibit the expression of annexin A3 in the cell, preferably wherein the annexin in the cell is reduced by using a DNA construct or an expression vector expressing antisense annexin A3 A3 content.

Therefore, the present invention also relates to agents that reduce and/or inhibit the expression of annexin A3 in cancer cells, such as antisense nucleotides that inhibit annexin A3 expression and ribozymes that specifically cut annexinA3 mRNA; Use of the drug in cancer resistant to platinum-based chemotherapy drugs. In one embodiment, according to the antisense nucleotide of the present invention, it comprises the nucleotide sequence of all or part of any sequence capable of specifically binding to the annexin A3 polynucleotide sequence or its complementary sequence, preferably the antisense core The nucleotide contains a sequence region that is complementary, more preferably substantially complementary, even more preferably fully complementary to one or more portions of the annexin A3 polynucleotide, and more preferably, the antisense nucleotide comprises a sequence region that is complementary to the full-length annexin The fully complementary DNA sequence of the A3 polynucleotide, preferably, the antisense nucleotide is DNA or RNA or derivatives thereof, most preferably, the antisense nucleotide is contained in an expression plasmid capable of expressing it, preferably in eukaryotic expression plasmids. In one embodiment, the ribozyme capable of specifically cleaving annexin A3 mRNA according to the present invention has a specific substrate binding site complementary to one or more annexin A3 RNA regions, and it has a substrate binding site in or around the substrate binding site The nucleotide sequence that confers this molecule RNA cutting activity, preferably said ribozyme is selected from: hammerhead type ribozyme, hairpin type ribozyme, hepatitis D virus type ribozyme, I type intron type ribozyme or RNaseP RNA-type ribozyme or Neurospora VS RNA-type ribozyme.

Another aspect of the present invention relates to cancer cells whose resistance to platinum-based chemotherapy drugs has been changed through the aforementioned method for changing cancer drug resistance of the present invention, preferably, the cells contain cells expressing sense annexin A3 or antisense annexin A3 An expression vector, preferably, the vector is a plasmid.

Another aspect of the present invention relates to a method for screening agents capable of changing the drug resistance of cancer to platinum-based chemotherapy drugs, wherein the cancer is preferably ovarian cancer, and the platinum-based chemotherapy drugs are preferably cisplatin or carboplatin, The method comprises the steps of: providing a candidate drug; contacting the candidate drug with cancer cells; detecting the expression of annexin A3 in the cancer cells; and comparing the detected expression amount with a predetermined threshold. Accordingly, the present invention also relates to agents also obtained by the screening method of the present invention.

One aspect of the present invention relates to a method for detecting drug resistance of cancer cells to platinum-based chemotherapy drugs, wherein the cancer is preferably ovarian cancer, and the platinum-based chemotherapy drugs are preferably cisplatin or carboplatin, the method It includes detecting the expression level of annexin A3 in cancer cells, and comparing the obtained expression level with a predetermined threshold, thereby indicating the resistance of ovarian cancer cells to platinum chemotherapy drugs.

Another aspect of the present invention relates to a method for predicting or prognosing the therapeutic effect of platinum-based chemotherapy drugs in cancer patients, that is, diagnosing the resistance of platinum-based chemotherapy drugs in cancer patients, wherein the cancer is preferably ovarian cancer, The platinum chemotherapeutic drug is preferably cisplatin, carboplatin, and the method includes detecting the expression level of annexin A3 in the tumor tissue of a cancer patient, and comparing the obtained amount with a predetermined threshold, thus predicting or prognosticating to the selected The therapeutic effect of platinum-based chemotherapeutic drugs administered to the above-mentioned patients, that is, to predict the patient's resistance to platinum-based chemotherapeutic drugs.

In the above method of the present invention, the step for detecting the expression of annexin A3 is by detecting the mRNA level or protein level of annexin A3; wherein, preferably, the detection at the protein level is by antibody or oligonucleotide aptamer To achieve, preferably using antibodies; and detection at the mRNA level, preferably by Northern blotting or PCR methods to achieve.

Correspondingly, the present invention also relates to agents capable of detecting the expression of annexin A3. In one embodiment, the detection agent is an antibody to annexin A3 or an antigen-binding fragment thereof, wherein the antibody can be a monoclonal antibody or a polyclonal antibody, preferably a neutralizing antibody. In another embodiment, the detection agent is an oligonucleotide aptamer specifically binding to annexin A3 protein, preferably the oligonucleotide aptamer is DNA, preferably, the oligonucleotide is suitable for Ligands are linked or labeled with other functional groups or molecules, such as sulfhydryl, amino, radioactive isotope, fluorescein, biotin, or enzymes. In yet another embodiment, the detection agent is a nucleic acid probe or primer or a combination thereof specific to the annexin A3 gene.

The present invention also relates to the use of the aforementioned agent capable of detecting the expression of annexin A3 in the preparation of a detection or diagnostic kit for detecting the resistance of cancer cells to platinum-based chemotherapy drugs or diagnosing the resistance of cancer patients to platinum-based chemotherapy drugs. For this purpose of the present invention, the medicament is preferably an antibody to the annexin A3 protein, preferably a monoclonal antibody; or the medicament detects the mRNA level expression of the annexin A3 gene in the cell, preferably a hybridization probe specific to the annexin A3 gene or primers.

Still another aspect of the present invention relates to a method for treating cancer patients, comprising the steps of: a) reducing the patient's tolerance to platinum-based chemotherapeutic drugs by using the method of the present invention for changing the resistance of cancer platinum-based chemotherapeutic drugs; The patients are administered platinum-based chemotherapy drugs, and optionally, the resistance of cancer patients to platinum-based chemotherapy drugs is detected before step (a) to determine drug-resistant patients.

Yet another aspect of the present invention relates to a method for treating a cancer patient, the method comprising: determining the patient's platinum-based chemotherapy drug resistance according to the present invention, and adjusting the patient's platinum-based chemotherapy drug dosing regimen. In this aspect of the invention, the adjustment includes substituting paclitaxel-based chemotherapy drugs, increasing or decreasing the dosing frequency and/or dosage of paclitaxel-based chemotherapy drugs, and the like.

Other aspects of the present invention also relate to a pharmaceutical composition, which comprises the aforementioned agent capable of changing the drug resistance of cancer of the present invention or the aforementioned agent capable of detecting the expression of annexin A3. In some embodiments of the present invention, the pharmaceutical composition comprises one or any combination of DNA expressing annexin A3, antisense nucleotides of annexin A3, ribozymes, antibodies and oligonucleotide aptamers. In some embodiments, the pharmaceutical composition of the present invention is used to diagnose and/or alter the resistance of a cancer to a platinum-based chemotherapy drug, wherein the cancer is ovarian cancer.

The last aspect of the present invention relates to a method for establishing platinum-based chemotherapeutic drug-resistant cancer cell lines, comprising: inducing cancer cells with a large dose of platinum-based chemotherapeutic drugs.

In all aspects of the invention, it is preferred, if applicable, that the cancer is a cancer that can be treated with platinum-based chemotherapeutics, such as ovarian cancer, breast cancer, lung cancer, testicular tumors, head and neck cancer, osteosarcoma, melanoma, Esophageal cancer, etc., preferably ovarian cancer, in one embodiment, preferably ovarian epithelial cancer, including endometrioid carcinoma, seromammary carcinoma, adenocarcinoma, transitional cell carcinoma, endometrial carcinoma, clear cell carcinoma, in another embodiment , preferably human serous ovarian carcinoma.

In all aspects of the present invention, if applicable, preferably, the platinum-based chemotherapeutic drug is cisplatin, carboplatin, oxalplatin, nedaplatin, roplatin, etc., more preferably cisplatin and carboplatin.

Brief description of attached drawings

Figure 1. Schematic diagram of CDDP high-dose shock induction method

Figure 2. Schematic diagram of CDDP small-dose intermittent induction method

Figure 3. Dose-response curves of CDDP to SKOV3, SKOV3/CDDP-P and SKOV3/CDDP-80

Figure 4. Light microscope observation of cell morphology (×200)

a.SKOV3; b.SKOV3/CDDP-P; c.SKOV3/CDDP-80; d, e, f are the cell morphology of a, b, c grown to logarithmic growth phase

Figure 5.a. From left to right, the cell morphology of SKOV3, SKOV3/CDDP-P and SKOV3/CDDP-80 under scanning electron microscope (×1600)

The SKOV3 cells were spindle-shaped, and the microvilli on the cell surface were abundant, long and slender, and evenly distributed. Both CDDP-P and CDDP-80 showed that the microvilli on the cell surface were sparse and unevenly distributed. The morphology of CDDP-80 changed significantly, and the cells extended from the nucleus to both sides with a larger area. b, c. From left to right, the cell morphology of SKOV3, SKOV3/CDDP-P and SKOV3/CDDP-80 under the transmission electron microscope (b, ×8000; c, ×22000). It can be seen that SKOV3 cytoplasm contains a large number of mitochondria, CDDP -P and CDDP-80 cells have large and irregular nuclei, deformed nuclei can be seen, nuclear membrane depressions are obvious, nuclear pockets and nuclear processes are present. The chromatin is unevenly distributed, some condense into blocks, and the mitochondria in the cytoplasm are reduced, with a large number of vesicle-like structures. Some of these vesicle-like structures are vacuoles, and some of them can be seen as granules.

Figure 6. Growth curves of SKOV3, SKOV3/CDDP-P and SKOV3/CDDP-80

Figure 7. RT-PCR determination of the expression of MDR1, MRP1, LRP1, GST-pi mRNA

^* P<0.01 compared with SKOV3

^** P<0.01 compared with SKOV3 and SKOV3/CDDP-80

Figure 8. Expression statistics of MDR1, MRP1, LRP1, GST-pi mRNA in different cell lines

^* P<0.01 compared with SKOV3

^** P<0.01 compared with SKOV3 and SKOV3/CDDP-80

Figure 9 Western blot method to determine the expression of MDR1, MRP1, LRP1, GST-pi protein

Figure 10. All differentially expressed protein spots of four drug-resistant cell lines

Figure 11. Expression of protein spot 25 (arrow) in 2-DE profiles of six cell lines, identified as annexin A3 by MALDI-TOF-MS

Figure 12. Peptide mass fingerprint of protein spot 25 (annexin A3) identified by MALDI-TOF-MS

Figure 13. Expression of protein spot 67 (at the arrow) in the 2-DE profiles of six cell lines, identified as destrin by MALDI-TOF-MS

Figure 14. Expression of protein spot 7 (arrow) in 2-DE profiles of six cell lines, identified as IDHc by MALDI-TOF-MS

Figure 15. Expression of protein spot 54 (arrow) in 2-DE profiles of six cell lines, identified as GSTO1-1 by MALDI-TOF-MS

Figure 16. Expression of protein spot 34 (at the arrow) in the 2-DE profiles of six cell lines, identified as cofilin 1 by MALDI-TOF-MS

Figure 17. Quantitative PCR method to detect the mRNA expression of five differentially expressed proteins in six cell lines

Figure 18. Western blot method to detect the expression of four differentially expressed proteins in six cell lines

Figure 19. pcDNA3.1/myc-His(-)B plasmid map

Figure 20. Schematic diagram of the construction process of pcDB-sense anx3 and pcDB-antisense anx3 plasmids

Figure 21. pcDNA3.1(+) plasmid map

Figure 22. Schematic diagram of the construction process of pcDNA3.1-anx3 plasmid

Figure 23. Plasmid map of pEGFP-N1

Figure 24. Schematic diagram of the pEGFP-N1-anx3 plasmid construction process

Figure 25. The electrophoresis image of total cellular RNA, 28, 16S, and 5S bands can be seen

Figure 26. Amplification of annexin A3 (anx 3) by RT-PCR

Lane 1 uses SKOV3 cell cDNA as a template to see a bright band at 1000bp

Figure 27. Identification of T recombinant plasmids by PCR and enzyme digestion

Lane 1: Using the pcDB-sense anx3 plasmid as a template, the expected band can be amplified at 1000bp

lane 2: EcoR I single enzyme digestion can cut out the target band

lane 3: Undigested T recombinant plasmid

Figure 28. Alignment results in Genebank after sequencing of the recombinant T plasmid (pGEM-T-anx3)

Figure 29. Identification of pcDB-sense anx3 and pcDB-antisense anx3 plasmids by PCR and enzyme digestion

lane 1: Negative control without upstream primers

Lane 2: Using the pcDB-sense anx3 plasmid as a template, the expected band can be amplified at 1000bp

Lane 3: Using the pcDB-antisense anx3 plasmid as a template, the expected band can be amplified at 1000bp

lane 4: undigested pcDB-antisense anx3 plasmid

Lane 5: pcDB-sense anx3 plasmid digested by EcoR I, which can cut out the target band

Lane 6: pcDB-antisense anx3 plasmid digested by EcoR I, which can cut out the target band

Figure 30. The alignment results of the pcDB-antisense anx3 plasmid in Genebank after sequencing

Figure 31.a. Identification of pcDNA3.1-anx3 plasmid by PCR method

Lane 1: Using the pcDNA3.1-anx3 plasmid as a template, the expected band can be amplified at 1300bp

b. Identification of pcDNA3.1-anx3 plasmid by enzyme digestion

lane1: EcoR I single enzyme digestion can cut out the target band

Figure 32. pcDNA3.1-anx3 plasmid alignment results in Genebank after sequencing

Figure 33. Identification of pEGFP-N1-anx3 plasmid by PCR method

Lane 1: The pEGFP-N1-anx3 plasmid is used as a template, and the expected band can be amplified at 1000bp

Figure 34. Identification of pEGFP-N1-anx3 plasmid by enzyme digestion

lane 1: BamH I single enzyme digestion without target band

Lane 2: Xho I, BamH I double enzyme digestion can see the target band

Figure 35. The alignment results of the pEGFP-N1-anx3 plasmid in Genebank after sequencing

Figure 36. pEGFP-N1-anx3 plasmids were transfected into A2780 and SKOV3 cells respectively, and the transfection efficiency observed under the fluorescence microscope (24h after transfection)

a, c: SKOV3 cells transfected by pEGFP-N1-anx3 plasmid. The transfection efficiency of vector pEGFP-N1-anx3 plasmid into SKOV3 cells is about 20-25% (×200);

b, d: A2780 cells transfected by pEGFP-N1-anx3 plasmid. The transfection efficiency of the vector pEGFP-N1-anx3 after transfecting A2780 cells for 24 hours was about 45-55% (×200)

Figure 37. Colony morphology of SKOV3 and A2780 cells during screening for stable transfection

Figure 38. Western blot identification results of stably transfected cell clones Sensitive annexin A3 plasmid transfects sensitive cell lines SKOV3 and A2780, antisense annexin A3 plasmid transfects drug-resistant cell lines SKOV3/CDDP, SKOV3/CBP, A2780/CDDP and A2780 /CBP

a.annexin A3; b.beta-actin; C. untransfected cell control; M. cell control transfected with corresponding empty plasmid; 1-7 are cell clones transfected with corresponding annexin A3 sense or antisense plasmid

Figure 39. Annexin A3 immunohistochemical staining results (×200)

a.Annexin A3 staining negative

b.Annexin A3 staining is positive, and the positive part is the nucleus

c, d.annexin A3 staining positive, the positive part is the cytoplasm

Detailed ways

This specification further elaborates on the present invention in the following aspects.

Annexin A3 protein and its coding nucleotide

The annexin A3 or annexin A3 protein referred to in the present invention includes homologues, allelic variants, functional equivalents of annexin A3, especially conservative variants, preferably refers to human annexinA3 protein. Annexin A3 protein conservative variants include proteins having one or more (e.g., 1-20, 1-10, 1-5) amino acid insertions, deletions, and/or additions compared to the annexin A3 proteins disclosed herein . In the present invention, "polypeptide" and "protein" can be used interchangeably, and can refer to polypeptides of any length.

Those skilled in the art will appreciate that polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond to DNA molecules in a one-to-one fashion, and mRNA molecules, which do not contain introns.

A polynucleotide may contain a native sequence (ie, an endogenous sequence encoding an annexin A3 protein or a portion thereof) or may contain a variant of this sequence, or a biological or functional equivalent. A polynucleotide variant may contain one or more substitutions, additions, deletions and/or insertions, preferably, these changes are conservative or such changes result in a polynucleotide variant that retains the ability to encode a protein of the invention.

When comparing polynucleotide or polypeptide sequences, two sequences are said to be "identical" if the nucleotide or amino acid sequences in the two sequences are identical when aligned for maximum matching as described below.

A preferred example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms described in Altschul et al. (1977) Nucl. Acid. Res. 25:3389-3402 and Altschul et al. (1990) respectively J. Mol. Biol. 215:403-410. Using parameters such as those described herein, BLAST and BLAST 2.0 can be used to determine percent sequence identity for polynucleotides and polypeptides of the invention. Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information.

Accordingly, the invention includes polynucleotide and polypeptide sequences substantially identical to the sequences disclosed herein, for example, when using the methods described herein (eg, BLAST analysis using standard parameters, described below), with polynucleotide or polypeptide sequences of the invention Contains at least 50% sequence identity, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% compared to or higher sequence identities. Those skilled in the art will appreciate that appropriate adjustments to these values may be made to determine the corresponding identity of proteins encoded by two nucleotide sequences to take into account codon degeneracy, amino acid similarity, positioning of reading frames, and the like.

In other embodiments, the invention provides isolated polynucleotides and polypeptides comprising contiguous stretches of various lengths that are identical or complementary to one or more of the sequences disclosed herein. For example, the invention provides at least about 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, or 1000 or more contiguous nucleosides comprising one or more of the sequences disclosed herein acids, and polynucleotides containing all intermediate length contiguous nucleotides in between. It will be readily apparent that "intermediate length" refers herein to any length between these quoted values, for example 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51 , 51, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers between 200-500, 500-1,000, etc.

The polynucleotides of the present invention, or fragments thereof, no matter how long their own coding sequences are, can be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites , other coding segments, etc., such that their overall lengths may vary considerably. Thus, it is contemplated that nucleotide fragments of virtually any length may be employed, the total length of which is preferably limited to that which permits ease of manipulation and application in desired recombinant DNA procedures. For example, exemplary DNA segments with a total length of about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs, etc. (including all intermediate lengths) are expected to be in are useful in many embodiments of the invention.

In other embodiments, the invention relates to polynucleotides capable of hybridizing to the polynucleotides provided herein, or fragments thereof, or complements thereof, under conditions of moderate stringency, preferably high stringency. Hybridization techniques are well known in the field of molecular biology.

Typically, "hybridization conditions" are classified according to the degree of "stringency" of the conditions under which hybridization is measured. The degree of stringency can be based on, for example, the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, "maximum stringency" typically occurs at about Tm-5°C (5°C below the Tm of the probe); "high stringency" occurs at about 5-10°C below the Tm; About 10-20°C below the Tm; "low stringency" occurs at about 20-25°C below the Tm. Alternatively, or in addition, hybridization conditions may be based on salt or ionic strength conditions for hybridization and/or one or more stringency washes. For example, 6 x SSC = very low stringency; 3 x SSC = low to medium stringency; 1 x SSC = medium stringency; 0.5 x SSC = high stringency. Functionally, conditions of maximum stringency can be used to determine nucleic acid sequences that are strictly identical or nearly identical to a hybridization probe; while conditions of higher stringency can be used to determine nucleic acid sequences that have about 80% or more sequence identity to the probe .

For applications requiring high selectivity, it is typically desirable to employ relatively stringent conditions for hybrid formation, eg, selecting relatively low salt and/or high temperature conditions. Sambrook et al. (Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.) provide hybridization conditions including medium stringency and high stringency.

For ease of illustration, suitable moderately stringent conditions for detecting the hybridization of polynucleotides of the present invention to other polynucleotides include: prewashing with 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) solution; Hybridization was performed overnight in 5X SSC at 50-65°C; followed by two washes each at 65°C for 20 minutes in 2X, 0.5X, and 0.2X SSC containing 0.1% SDS. Those skilled in the art will appreciate that the stringency of hybridization can be easily manipulated, such as changing the salt content of the hybridization solution and/or the hybridization temperature. For example, in another embodiment, suitable highly stringent hybridization conditions include the conditions described above, except that the hybridization temperature is increased to, for example, 60-65°C or 65-70°C.

The polynucleotides and polypeptides of the present invention can be prepared according to methods known in the art. See eg common laboratory handbook.

change in expression

One aspect of the present invention relates to modulating the resistance of cancer to platinum-based chemotherapy drugs by altering the expression of annexin A3 in cancer cells.

In this regard, said alteration of expression includes up-regulation and/or activation of expression thereby increasing the resistance of cancer to platinum-based chemotherapy drugs, and down-regulation and/or inhibition of expression thereby reducing cancer resistance to platinum-based chemotherapy drugs Medicinal properties. The modulation of the drug resistance of cancer according to the present invention can be administered to cancer cells in vitro, for example to obtain drug-resistant cell lines or reduce the drug resistance of formerly drug-resistant cells. Modulation of drug resistance of cancer according to the invention can also occur in vivo in a patient suffering from said cancer to alter, preferably reduce, the patient's resistance to platinum-based chemotherapeutic drugs.

As used herein, "upregulation of expression" or "activation of expression" refers to increasing the level of gene expression and/or the level of active gene product and/or the level of gene product activity. For example, it can be achieved by expressing exogenous annexin A3 in cancer cells, enhancing the expression of endogenous annexin A3 in cancer cells, and the like. In the present invention, exogenous annexin A3 refers to annexin A3 introduced by genetic engineering technology; and endogenous annexin A3 refers to the original annexinA3 in cells. Any method known to those skilled in the art that can increase the level of expression of the gene of interest and/or the level of active gene product and/or the level of activity of the gene product is applicable to this aspect of the present invention and is therefore also included within the scope of the present invention .

As used herein, "downregulation of expression" or "inhibition of expression" refers to reducing the level of gene expression and/or the level of active gene product and/or the level of gene product activity. As described by Angell and Baulcombe (1998-WO9836083), Lowe et al., (1989-WO9853083), Lederer et al., (1999-WO9915682) or Wang et al. (1999-WO9953050), by, for example, in a sense relative to the promoter sequence Coding sequences or parts thereof can be added in orientation (if cosuppression is caused) or in antisense orientation, and reduction of expression can be accomplished, for example, by insertional mutagenesis (eg T-DNA insertion or transposon insertion) or by gene silencing strategies. Gene constructs aimed at silencing gene expression may have the nucleotide sequence (or one or more parts thereof) of said gene contained in sense and/or antisense orientation relative to a promoter sequence. Another approach to downregulate gene expression involves the use of ribozymes.

Modulation, including reducing the level of an active gene product or gene product activity, may be effected by administering to or exposing a cell, tissue, organ or organism to said compound or agent of the invention.

Immunomodulation is an example of another technique having the ability to down-regulate an active gene product and/or the level of gene product activity comprising administering or exposing antibodies to said gene product wherein the level of said gene product and/or gene product activity is to be modulated cells, tissues, organs or organisms, or antibodies expressing the gene product in such cells, tissues, organs or organisms. Such antibodies include single chain antibodies, IgG antibodies and fragments thereof.

Modulation, including enhancing/decreasing the level of an active gene product or gene product activity, can also be achieved by administering to or exposing the cell, tissue, organ or organism to an agonist/antagonist of the gene product or its activity. Such agonists/antagonists include proteins and chemical compounds identified according to the invention as described.

Upregulation/downregulation of the expression of the annexin A3 gene is envisaged in the context of the present invention. The present invention also includes up-regulation/down-regulation of annexin A3 activity level.

In addition, gene silencing by recombination can be considered.

"Recombinase" means a site-specific recombinase or transposase. "Recombination site" means a site-specific recombination site or a transposon border sequence. "Site-specific recombination event" means an event catalyzed by a system generally consisting of 3 elements: a pair of DNA sequences (site-specific recombination sequence or site) and a specific enzyme (site-specific recombinase) . Site-specific recombinases only catalyze recombination reactions between two site-specific recombination sequences according to the orientation of the site-specific recombination sequences. When the site-specific recombination sequences are oriented in opposite directions (i.e., inverted repeats) in the presence of a site-specific recombinase, sequences intervening between two site-specific recombination sites will inverted. If the site-specific sequences are oriented in the same orientation as each other (ie, direct repeats), any intervening sequences will be lost upon interaction with the site-specific recombinase. Therefore, if the site-specific recombination sequence exists as a direct repeat at both ends of the foreign DNA integrated into the eukaryotic genome, this integration of said sequence can then be replaced by the site-specific recombination sequence with the corresponding The interaction of the site-specific recombinase is reversed.

A number of different site-specific recombinase systems are available, including but not limited to the Cre/lox system of bacteriophage P1, the FLP/FRT system of yeast, the Gin recombinase of bacteriophage Mu, and the Pin recombinase of E. coli , PinB, PinD and PinF of Shigella, and the R/RS system of the pSR1 plasmid. Recombinases are usually integrases, resolvases or flippases. Furthermore, the bispecific recombinase can be used in combination with direct or indirect repeats corresponding to two different site-specific recombination sites of the bispecific recombinase (WO99/25840). Two preferred site-specific recombinase systems are the bacteriophage P1 Cre/lox and the yeast FLP/FRT system. In these systems, a recombinase (Cre or FLP) interacts with its respective site-specific recombination sequence (lox or FRT, respectively) to invert or excise intervening sequences.

Although site-specific recombination sequences must be ligated to the ends of the DNA to be excised or inverted, the gene encoding the site-specific recombinase can be located elsewhere. For example, the recombinase gene may already be present in the eukaryotic DNA or may be provided by a later introduced DNA fragment, which may be introduced into the cell by crossover or by heteropollination or directly. As an alternative, substantially purified recombinant enzyme protein can be introduced directly into eukaryotic cells, for example, by microinjection or particle bombardment. Typically, the site-specific recombinase coding region will be operably linked to regulatory sequences that enable expression of the site-specific recombinase in eukaryotic cells.

In carrying out the invention, it is preferred that the genetic element be completely excised at the original integration site or alternatively be left, for example by expression of a recombinase protein in the cell which contacts the integration site of the genetic element and facilitates a recombination event therein. A "footprint", typically about 20 nucleotides in length or longer, is used to induce movement of genetic elements. These hosts and host portions produced according to the methods of the invention can be identified by standard nucleic acid hybridization and/or amplification techniques to detect the presence of mobile genetic elements or genetic constructs comprising them. Alternatively, in the case of transformed host cells, tissues and hosts in which the mobile genetic element has been excised, this technique can be used to detect the footprint in the host genome left after the excision event. The term "footprint" as used herein should be understood as referring to any derivative of the mobile genetic element or genetic construct containing the element described herein, by excision of the mobile genetic element in the genome of a cell transformed with the genetic construct previously described, Deletion or other removal can produce it. Typically, the footprint contains at least a single copy of a transposon or recombination site for facilitating excision. However, the footprint may contain other sequences derived from the genetic construct, for example, if used, a nucleotide sequence derived from a left border sequence, a right border sequence, an origin of replication, a recombinase coding sequence or a transposase coding sequence, or other Nucleotide sequence of vector origin. Thus, footprints can be identified based on the nucleotide sequence of the recombinant locus or transposon of the genetic construct used, such as, for example, a nucleotide sequence corresponding to or complementary to a lox site or frt site.

The present invention also relates to the inhibition of annexin A3 in vitro and in vivo by using antisense technology. Antisense technology can be used to control gene expression through triple helix formation or through antisense DNA or RNA, both of which are based on the binding of polynucleotides to DNA or RNA. For example, the 5' coding portion of a polynucleotide encoding a polypeptide of the invention is used to design antisense RNA nucleotides about 10-40 base pairs in length. Design DNA nucleotides complementary to regions involved in transcription in the gene (triple-stranded helix see Lee et al., Nucl. Acids Res., 6:3073, 1979; Cooney et al., Science, 241:456, 1988; and Dervan et al. People, Science, 251:1360, 1991), thereby preventing the transcription and production of annexin A3. Antisense RNA nucleotides hybridize with mRNA in vivo, and block the translation of mRNA molecules into annexin A3 protein (antisense participates in Okano, J.Neurochem., 56:560, 1991; "Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression" (oligonucleotides) Nucleotides as Antisense Inhibitors of Gene Expression), CRC Press, Boca Raton, FL, 1988).

Alternatively, the above-mentioned nucleotides can be delivered to cells by procedures in the art, so that the antisense RNA or DNA can be expressed in vivo, thereby inhibiting the production of annexinA3 in the above-mentioned manner.

antisense nucleotide

The end result of the flow of genetic information is the synthesis of proteins. DNA is transcribed into messenger RNA by polymerases, which is then translated on ribosomes to produce folded, functional proteins. Thus, there are several steps along this pathway at which inhibition of protein synthesis can be achieved. Native DNA segments encoding polypeptides described herein, like all such mammalian DNA strands, have two strands held together by hydrogen bonding: a sense strand and an antisense strand. The messenger RNA encoding the polypeptide has the same nucleotide sequence as the sense DNA strand, except that thymidine in the DNA is replaced by uridine. Thus, the synthetic antisense nucleotide sequence will bind to the mRNA and inhibit the expression of the protein encoded by the mRNA.

Targeting antisense nucleotides to mRNA is therefore a mechanism for shutting down protein synthesis and thus a powerful targeted therapy. For example, the synthesis of polygalacturonase and muscarinic acetylcholine receptor type 2 is inhibited by antisense nucleotides directed to their respective mRNA sequences (US Patent 5,739,119 and US Patent 5,759,829, both of which are hereby fully incorporated herein by reference). Furthermore, cell cycle nucleoproteins, broad-spectrum drug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABAA receptor and human EGF (Jaskulski et al., 1988; Vasanthakumar and Ahmed, 1989 Peris et al., 1998; US Patent 5,801,154; US Patent 5,789,573; US Patent 5,718,709 and US Patent 5,610,288, all of which are hereby incorporated by reference in their entirety) also show examples of antisense suppression.

Accordingly, in exemplary embodiments, the invention provides nucleotide sequences comprising all or part of any sequence capable of specifically binding to a polynucleotide sequence described herein or its complement. In one embodiment, the antisense nucleotides comprise DNA or derivatives thereof. In another embodiment, the nucleotide comprises RNA or a derivative thereof. In a third embodiment, the nucleotide is a modified DNA containing a phosphorothioate modified backbone. In a fourth embodiment, the nucleotide sequence comprises a peptide nucleic acid or a derivative thereof. In all cases, preferred compositions contain sequence regions that are partially complementary, more preferably substantially complementary, even more preferably fully complementary to one or more portions of the polynucleotides disclosed herein. The one or more portions may be about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, or 1000 or more contiguous nucleotides, and All consecutive nucleotides of intermediate length between. It will be readily apparent that "intermediate length" refers herein to any length between these quoted values, for example 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51 , 51, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers between 200-500, 500-1,000, etc. Thus, those skilled in the art will understand that the nucleotides of the present invention can have as few as about 10 and as many as about 1000 or more nucleotides.

Selection of antisense compositions specific for a given gene sequence is based on analysis of the selected target sequence (e.g., human sequence in an illustrative example), and determination of secondary structure, Tm, binding energy, relative stability. are performed, and antisense compositions are also selected based on their relative lack of ability to form dimers, hairpins, or other secondary structures that would reduce or prevent specific binding to the target mRNA in the host cell.

Highly preferred target regions in mRNA are those located at or near the AUG translation initiation codon, and those sequences that are substantially complementary to the 5' region of the mRNA. Analysis of these secondary structures and consideration of the selection of target sites was carried out using the OLIGO primer analysis software version 4 (Rychlik, 1997) and the BLASTN 2.0.5 algorithm software (Altschul et al., 1997).

The inventors also contemplated using an antisense delivery method via a short peptide carrier called MPG (27 residues). The MPG peptide contains a hydrophobic domain derived from the HIV gp41 fusion sequence and a hydrophilic domain derived from the SV40 T antigen nuclear localization sequence (Morris et al., 1997). It has been demonstrated that a few MPG peptide molecules can be coated with antisense nucleotides and that they can be delivered with relatively high efficiency (90%) into cultured mammalian cells in less than 1 hour. Furthermore, interaction with MPG greatly increases the stability of this nucleotide against nucleases and the ability to cross the plasma membrane (Morris et al., 1997).

ribozyme

Although proteins have traditionally been used to catalyze nucleic acid reactions, another class of macromolecules has been shown to be useful in this regard. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific manner. Ribozymes possess a specific catalytic domain that possesses endonuclease activity (Kim and Cech, 1987; Gerlach et al., 1987; Forster and Symons, 1987). For example, many ribozymes accelerate phosphate transfer reactions with a high degree of specificity, often cleaving only one of several phosphate esters of oligonucleotide substrates (Cech et al., 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub , 1992). This specificity is attributed to the requirement that the substrate bind via specific base-pairing interactions to the ribozyme's internal guide sequence ("IGS") prior to the chemical reaction.

Initially, reactions catalyzed by ribozymes were observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cech et al. 1981). For example, U.S. Pat. No. 5,354,855 (hereby incorporated by reference) reports that certain ribozymes are capable of functioning as endonucleases with a sequence specificity higher than that of known ribozymes and approaching that of Sequence specificity of DNA restriction enzymes. Therefore, sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suitable for therapeutic applications (Scanlon et al., 1991; Sarver et al., 1990). Recently, it has been reported that ribozymes induce genetic changes in some cell lines to which they have been applied; these altered genes include genes for the oncogenes H-ras, c-fos, and HIV. Much of this work involves the modification of target mRNAs based on the cleavage of specific mutant codons by specific ribozymes.

Six basic variants of naturally occurring enzymatic RNAs are currently known. Each is capable of catalyzing the hydrolysis of RNA phosphodiester bonds (and thus the ability to cleave other RNA molecules) in trans under physiological conditions. Typically, an enzymatic nucleic acid acts by first binding to a target RNA. This binding occurs through the target binding portion of the enzymatic nucleic acid, which is in close proximity to the enzymatic portion of the molecule that cleaves the target RNA. Thus, the enzymatic nucleic acid first recognizes through complementary base pairing, then binds to the target RNA, and once bound to the correct site, enzymatically cleaves the target RNA. Strategic cleavage of this target RNA will destroy its ability to direct the synthesis of the encoded protein. After the enzymatic nucleic acid binds and cleaves its RNA target, it is released from that RNA to seek another target and can repeatedly bind and cleave the new target.

The enzymatic properties of ribozymes are superior to many technologies, such as antisense technology (in which a nucleic acid molecule simply binds to a nucleic acid target to block its translation), because the concentration of ribozyme necessary to achieve therapeutic treatment is greater than that required Low concentration of antisense oligonucleotides. This advantage reflects the ability of ribozymes to act enzymatically. Thus, a single ribozyme molecule is capable of cleaving many target RNA molecules. In addition, ribozyme is also a highly specific inhibitor, and its inhibitory specificity depends not only on the base pairing mechanism of binding to target RNA, but also on the mechanism of cleavage of target RNA. A single base mismatch, or base substitution, near the cleavage site can completely abolish the catalytic activity of the ribozyme. Similar mismatches in antisense molecules do not prevent their action (Woolf et al., 1992). Thus, the specificity of action of ribozymes is higher than that of antisense oligonucleotides that bind to the same RNA site.

Enzymatic nucleic acid molecules can be formed in the motifs of hammerhead, hairpin, HDV, type I introns, or RNaseP RNA (associated with the RNA guide sequence) or Neurospora VS RNA. An example of a hammerhead motif is described in Rossi et al. (1992). Examples of hairpin motifs are described in Hampel et al. (European Patent Application Publication EP 0360257), Hampel and Tritz (1989), Hampel et al. (1990) and US Patent 5,631,359 (hereby incorporated herein by reference). Examples of hepatitis D virus motifs are described in Perrotta and Been (1992); examples of RNaseP motifs are described in Guerrier-Takada et al. (1983); ribozyme motifs for Neurospora VS RNA are described in Collins (Saville and Collins , 1990; Saville and Collins, 1991; Collins and Olive, 1993); examples of type I introns are described in US Patent 4,987,071 (hereby incorporated herein by reference). For the enzymatic nucleic acid molecule of the present invention it is only important that the molecule has a specific substrate binding site complementary to the RNA region of one or more target genes and that it has in or around the substrate binding site endowed to the molecule Nucleotide sequence for RNA cleavage activity. Therefore, there is no need to limit the structure of the ribozyme to the specific motifs mentioned herein.

In certain embodiments, it may be important to prepare enzymatic cleavage agents that exhibit high specificity for RNA of a target of interest, such as one of the sequences disclosed herein. The enzymatic nucleic acid molecule is preferably directed to a highly conserved sequence region of the target mRNA. These enzymatic nucleic acid molecules foreign to specific cells can be delivered as needed. Alternatively, ribozymes can be expressed from DNA or RNA vectors that are delivered to specific cells.

Small enzymatic nucleic acid motifs (such as hammerhead or hairpin motifs) can also be used for foreign delivery. The simple structure of these molecules increases the ability of the enzymatic nucleic acid to invade the target region of the mRNA structure. Alternatively, catalytic RNA molecules can be expressed in cells from eukaryotic promoters (e.g. Scanlon et al., 1991; Kashani-Sabet et al., 1992; Dropulic et al., 1992; Weerasinghe et al., 1991; Ojwang et al., 1992; Chen et al., 1992; Sarver et al., 1990). Those skilled in the art will appreciate that any ribozyme can be expressed in eukaryotic cells from a suitable DNA vector. The activity of these ribozymes can be increased by their release from the primary transcript by means of a second ribozyme (International Patent Application Publication WO 93/23569, and International Patent Application Publication WO 94/02595, both by which Incorporated herein by reference; Ohkawa et al., 1992; Taira et al., 1991; and Ventura et al., 1993).

Ribozymes can be added directly to target cells, or can be mixed with cationic lipids, lipid complexes, or packaged in liposomes, or otherwise delivered to target cells. The RNA or RNA complex can be locally administered ex vivo or in vivo to the relevant tissue by injection, aerosol inhalation, infusion pump or stent, with or without incorporation into a biopolymer.

Ribozymes can be designed and synthesized as described for in vitro and in vivo experiments as described in International Patent Application Publications WO 93/23569 and WO 94/02595 (both incorporated herein by reference). These ribozymes can also be optimized for delivery. Although specific examples are provided, those skilled in the art will appreciate that equivalent RNA targets in other species can be employed if desired.

Hammerhead or hairpin ribozymes can be analyzed individually by in silico folding (Jaeger et al., 1989) to assess whether the ribozyme sequence folds into the proper secondary structure. Those ribozymes with unfavorable intermolecular interactions between the binding arm and the catalytic core were excluded from consideration. Different binding arm lengths can be chosen to optimize activity. Typically, at least 5 or so bases on each arm are capable of binding to, or otherwise interacting with, the target RNA.

Ribozymes with hammerhead or hairpin motifs can be designed to anneal to various sites in the mRNA message and can be chemically synthesized. The synthesis method used is the same as the procedures described by Usman et al. (1987) and Scaringe et al. (1990) for normal RNA synthesis, and employs usual nucleic acid protecting and coupling groups, such as dimethoxytriphenylene at the 5' end methyl group, and a phosphoramidite at the 3' end. Typically, the average stepwise coupling yield is greater than 98%. Hairpin ribozymes can be synthesized in two parts, which are then annealed to reconstitute the active ribozyme (Chowrira and Burke, 1992). Ribozymes can be extensively modified so that by employing nuclease resistant groups such as 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-o-methyl, 2'-H Increased stability (for review see eg Usman and Cedergren, 1992). Ribozymes can be purified by gel electrophoresis or by high pressure liquid chromatography and resuspended in water using conventional methods.

Ribozyme activity can be optimized by altering the length of the ribozyme binding arm, or chemically synthesizing a modified ribozyme with modifications that protect the ribozyme from degradation by serum ribonucleases (see, e.g., International Patent Application Publication WO 92 /07065; Perrault et al., 1990; Pieken et al., 1991; Usman and Cedergren, 1992; International Patent Application Publication WO 93/15187; International Patent Application Publication WO 91/03162; European Patent Application Publication 92110298.4; and International Patent Application Publication WO 94/13688, which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules), modifications that enhance the effectiveness of ribozymes in cells, and modifications to shorten RNA synthesis time and reduce Scavenging of stem II bases by chemical necessity.

Sullivan et al. (International Patent Application Publication WO 94/02595) describe a general method for the delivery of enzymatic RNA molecules. Ribozymes can be administered to cells by a variety of methods known to those skilled in the art. These methods include, but are not limited to, encapsulation within liposomes, iontophoresis, or incorporation into other carriers such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For some cases, ribozymes can be delivered directly to cells or tissues ex vivo, with or without the vectors described above. Alternatively, local delivery of the RNA/vector combination can be performed by direct inhalation, direct injection, or using catheters, infusion pumps, or stents. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ophthalmic, intraperitoneal and/or intrathecal delivery . For a more detailed description of ribozyme delivery and administration see International Patent Application Publication WO 94/02595 and International Patent Application Publication WO 93/23569, all of which are hereby incorporated by reference.

Another method for accumulating high concentrations of ribozymes in cells is to incorporate the ribozyme coding sequence into a DNA expression vector. Transcription of the ribozyme sequence is driven by a promoter directed against eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts obtained from pol II or pol III promoters will be expressed at high levels in all cells; the level of a given pol II promoter in a given cell type will depend on the presence of nearby gene regulatory sequences (adders, silencers, etc. ) properties. Prokaryotic RNA polymerase promoters can also be used, as long as the prokaryotic RNA polymerase is expressed in appropriate cells (Elroy-Stein and Moss, 1990; Gao and Huang, 1993; Lieber et al., 1993; Zhou et al., 1990) . Ribozymes expressed from these promoters can function in mammalian cells (e.g. Kashani-Saber et al., 1992; Ojwang et al., 1992; Chen et al., 1992; Yu et al., 1993; L'Huillier et al., 1992; Lisziewicz et al., 1993). These transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including, but not limited to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors ( eg Retrovirus, Anonymous forest virus, Sindbis virus vector).

Ribozymes can be used as diagnostic tools to check for genetic drift and mutations in diseased cells. They can also be used to assess levels of target RNA molecules. The close relationship between ribozyme activity and target RNA structure allows detection of mutations in any region of the molecule that alters the base pairing and three-dimensional structure of the target RNA. Nucleotide changes important to RNA structure and function can be mapped in vitro, as well as in cells and tissues, by employing a variety of ribozymes. Cleavage of target RNA by ribozymes can be used to inhibit gene expression and (essentially) determine the role of a given gene product in disease progression. In this way, other genetic targets can be identified as important mediators of the disease. These studies will provide comprehensive treatment (such as multiple ribozymes targeting different genes, ribozyme conjugated with known small molecule inhibitors, or intermittent therapy combined with ribozymes and/or other chemical or biological molecules). Potential to lead to better treatment of the disease process. Other in vitro applications of ribozymes are well known in the art, including detection of the presence of mRNA associated with IL-5-associated diseases. The RNA is detected by assaying for the presence of the cleavage product following treatment with the ribozyme using standard methods.

Binding agent

The present invention also provides agents that specifically bind to the annexin A3 protein, such as antibodies and antigen-binding fragments thereof. In this context, an antibody or antigen-binding fragment thereof is considered to be related to annexin if it reacts (in, for example, in an ELISA) with the annexinA3 protein at a detectable level, whereas under similar conditions the reactivity with an unrelated protein cannot be detected. The A3 protein "specifically binds". As used herein, "associate" refers to a non-covalent association between two separate molecules such that a complex is formed. Binding ability can be evaluated, for example, by determining the binding constant for the formation of the complex. The binding constant is the value obtained when dividing the concentration of the complex by the product of the concentrations of the respective components. Generally, in the context of the present invention, two compounds are considered "associated" when the association constant for forming the complex is greater than about ¹⁰³ L/mol. The binding constant can be determined using methods well known in the art.

Using the representative assays provided herein, the binding agents are also capable of distinguishing whether a cancer patient/cancer cell is resistant to a platinum-based chemotherapeutic agent. In other words, an antibody or other binding agent that binds to the annexin A3 protein will produce a signal indicative of the presence of drug resistance in at least about 20% of drug-resistant patients/cancer cells and will produce a signal in at least about 90% of sensitive patients/cancer cells A signal indicating the absence of drug resistance. In order to determine whether a binding agent meets this requirement, the presence of the drug in biological samples (such as tumor biopsies) from drug-resistant and sensitive patients (determined by standard clinical tests) or in drug-resistant or sensitive cancer cells can be assayed as described herein. Whether there is a polypeptide bound to the binding agent in the Clearly, the number of resistant and sensitive samples analyzed should be statistically significant. Each binding agent should meet the above criteria; however, one of ordinary skill in the art will recognize that multiple binding agents can be used in combination to increase sensitivity.

Any agent that meets the above requirements can be a binding agent. For example, a binding agent can be a ribosome, RNA molecule or polypeptide with or without a peptide component. In a preferred embodiment, the binding agent is an antibody or antigen-binding fragment thereof. Antibodies can be prepared using any of a variety of techniques known to those of ordinary skill in the art. See eg Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, antibodies can be produced by cell culture techniques, including the production of monoclonal antibodies as described herein, or by transfecting a suitable bacterial or mammalian cell host with the antibody genes so as to allow recombinant antibody production. In one technique, any of a variety of mammals (eg, mice, rats, rabbits, sheep or goats) is first injected with an immunogen comprising a polypeptide of the invention. In this step, the polypeptide of the present invention can be used as an immunogen without modification. Alternatively, especially for relatively short polypeptides, an excellent immune response can be elicited if the polypeptide is linked to a carrier protein such as bovine serum albumin or keyhole limpet hemocyanin. The animal host is injected with the immunogen, preferably incorporating one or more booster immunizations according to a predetermined schedule, after which the animal is periodically bled. Polyclonal antibodies specific for the polypeptide can then be purified from the antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for the target antigen polypeptide can be prepared using, for example, the technique of Kohler and Milstein (Eur. J. Immunol. 6:511-519, 1976), and its improved method. Briefly, these methods involve the preparation of immortalized cell lines capable of producing antibodies with the desired specificity (ie, reactivity with a polypeptide of interest). These cell lines can be prepared, for example, from spleen cells obtained from animals immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably a myeloma cell that is isogenic to the immunized animal. A variety of fusion techniques can be employed. For example, the splenocytes and myeloma cells can be mixed with a non-ionic detergent for several minutes and then plated at low density on selective medium that supports the growth of hybrid cells but not myeloma cells. A preferred screening technique employs HAT (hypoxanthine, aminopterin, thymidine) screening. After a sufficient period of time, usually about 1-2 weeks, hybrid colonies can be observed. Single colonies were selected and their culture supernatants were tested for binding activity to the polypeptides of the invention. Hybridomas with high reactivity and specificity are preferred.

Monoclonal antibodies can be isolated from the supernatant of cultured hybridoma colonies. In addition, various techniques can be employed to increase yield, such as intraperitoneal injection of the hybridoma cell line into a suitable vertebrate host, such as a mouse. Monoclonal antibodies can then be harvested from ascitic fluid or blood. Impurities can be removed from these antibodies by conventional techniques such as chromatography, gel filtration, precipitation and extraction. Polypeptides of the invention may be used, for example, in affinity chromatography steps of purification methods.

In certain embodiments, it may be preferred to employ antigen-binding fragments of antibodies. These fragments include Fab fragments, which can be prepared using standard techniques. Briefly, immunoglobulins can be purified from rabbit serum by affinity chromatography on protein A bead columns (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988), followed by papain digestion Fab and Fc fragments are generated. The Fab and Fc fragments can be separated by affinity chromatography on a protein A bead column.

Monoclonal antibodies of the invention can be conjugated to one or more therapeutic agents. Suitable therapeutic agents in this regard include radionuclides, differentiation inducers, drugs, toxins and their derivatives. Preferred radionuclides ^include90Y , ^123I , ^125I , ^131I , ^186Re , ^188Re , ^{211At and212Bi} . Preferred drugs include methotrexate, and pyrimidine and purine analogs. Preferred differentiation inducers include phorbol esters and butyric acid. Preferred toxins include ricin, abrin, diphtheria toxin, cholera toxin, gelonin, pseudomonas exotoxin, shigella toxin and pokeweed antiviral protein.

oligonucleotide aptamer

SELEX technology is a new combinatorial chemical technology developed in recent years to study the structure, function and evolution of nucleic acids. It not only has good applications in basic research and biological screening, but also shows broad potential in clinical diagnosis. Prospects (Brody EN & Gold L, J. Biotechnol., 2000, 75:5-13). The screened oligonucleotide aptamers (aptamers) can recognize different molecules with high affinity and specificity. Its affinity and specificity are comparable to those of antibodies, and it is considered to be a new type of reagent that "challenges" the status of antibodies, and it plays an increasingly important role in the diagnosis and treatment of diseases. SELEX technology generally requires several rounds or dozens of rounds to obtain its corresponding ligand. Recently, Santa-Coloma TA et al reported highly specific "polyclonal oligobody (polyclonal oligonucleotide aptamer)", "monoclonal oligonucleotide (monoclonal oligonucleotide aptamer)" or "Synthetic oligobody (synthetic oligonucleotide aptamer)" (BianchiniM et al, J.Immunol.methods, 2001, 252: 191-197) can specifically recognize the corresponding protein, and is applied to Western blot, immunohistochemistry , Co-immunoprecipitation and other analysis experiments.

Screening for agents that modulate resistance to platinum chemotherapeutic drugs

The compound to be obtained or identified in this screening method of the present invention may be a compound capable of binding any nucleic acid, peptide or protein of the present invention. Other compounds of interest to be identified are compounds that modulate the expression of a gene or protein of the invention such that expression of the gene or protein is increased or decreased by the action of the compound. Alternatively, the compound may exert its effect by increasing or decreasing the activity of any protein of the invention.

The compound or the compounds may be comprised, for example, in a sample, such as a cell extract derived from an animal. Furthermore, said compounds may be known in the art, but so far are not known to have the ability to inhibit or activate annexin A3 interacting proteins. The reaction mixture can be a cell-free extract or can comprise cell or tissue culture. Suitable equipment for use in the methods of the invention are known to those skilled in the art and, in general, are described in Alberts et al., Molecular Biology of the Cell, 3rd Edition (1994), especially Chapter 17. Various compounds can, for example, be added to reaction mixtures, cell culture media or injected into cells.

If a sample containing a compound or compounds is identified in the method of the invention, the compound can be isolated from the original sample identified as containing a compound capable of acting as an antagonist/agonist, or if, for example, the original sample consists of a plurality of different compounds , one can further subdivide the original sample to reduce the number of different species per sample, and repeat the method with subdivided parts of the original sample. Depending on the complexity of the sample, the above steps can be carried out several times, preferably until the sample identified according to the method of the invention contains only a limited amount or only one substance. Preferably, said sample contains substances with similar chemical and/or physical properties, most preferably said substances are identical. Preferably, a compound or derivative thereof identified according to the methods described above is further formulated into a form suitable for administration to animals.

As a candidate agent for the method of the present invention, suitable computer programs can be used to carry out folding simulation and computer redesign of protein structural motifs of the present invention (Olszewski, Proteins 25 (1996), 286-299; Hoffman, Comput.Appl.Biosci. 1 (1995), 675-679) or can be obtained by screening compound combinatorial libraries. Computer simulations of protein folding can be used for conformational and energetic analysis of detailed peptide and protein models (Monge, J. Mol. Biol. 247 (1995), 995-1012; Renouf, Adv. Exp. Med. Biol. 376 (1995) , 37-45). In particular, suitable programs can be used for the identification of interaction sites of annexin A3/phosphorylated annexin A3, its ligands, or other interacting proteins by computer-aided retrieval of complementary peptide sequences (Fassina, Immunomethods 5 (1994), 114 -120). Moreover, in the background art, for example in Berry, Biochem.Soc.Trans.22 (1994), 1033-1036; Wodak, Ann, N.Y.Acac.Sci.501 (1987), 1-13; Pabo, Biochemistry 25 (1986 ), 5987-5991 describe suitable computer systems for protein and peptide design. The results obtained from the computer analysis described above can be used, for example, in the preparation of peptidomimetics of the proteins or fragments thereof of the invention. Pseudopeptide analogues of the native amino acid sequence of such proteins mimic the parent protein very effectively (Benkirane, J. Biol. Chem. 271 (1996), 33218-33224). For example, the incorporation of readily available achiral omega-amino acid residues in proteins or fragments thereof will result in the displacement of amino bonds by polymethylene units of aliphatic chains, thus providing a convenient strategy for constructing peptidomimetics (Banerjee , Biopolymers 39 (1996), 769-777). Hyperactive peptidomimetic analogs of small peptide hormones in other systems have also been described in the prior art (Zhang, Biochem. Biophys. Res. Commun. 224 (1996), 327-331). Combinatorial libraries of peptidomimetics can also be synthesized by sequential amine alkylation, and testing of compounds obtained therefrom, eg, for their binding, kinase inhibitory and/or immunological properties, to identify suitable peptidomimetics of the proteins of the invention. Methods and uses for the generation of combinatorial libraries of peptidomimetics are described in the prior art, for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715. Furthermore, the three-dimensional and/or crystal structure of the proteins of the invention can be used to design bioactive peptidomimetic inhibitors (Rose, Biochemistry 35 (1996), 12933-12944; Ruterber, Bioorg. Med. Chem. 4 (1996), 1545- 1558).

Correspondingly, the present invention also provides the medicament for regulating the drug resistance of the present invention, the pharmaceutical composition containing the medicament and the use of the medicament in preparing and regulating the drug resistance of the present invention.

Detection and diagnosis of drug resistance

Generally, based on the content of annexin A3 protein and/or the content of polynucleotides encoding these proteins in cancer cell lines or tumor biopsies obtained from patients, it can be detected whether the cancer cells and patients have resistance to platinum-based chemotherapy drugs sex. In general, the binding agents provided herein can detect the level of antigen bound to the agent in a biological sample. Polynucleotide primers and probes can be used to detect levels of mRNA encoding tumor proteins, which are also indicative of the presence or absence of drug resistance.

For detecting a polypeptide marker in a sample using a binding agent, a variety of assay formats known to those of ordinary skill in the art can be used. See, eg, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, resistance to platinum chemotherapy drugs in cancer cell lines and cancer patients can be determined by (a) contacting a cancer cell line or a tumor sample obtained from a patient with a binding agent; (b) detecting the presence of the binding agent in the sample. the level of bound polypeptide; and (c) comparing the polypeptide level to a predetermined cutoff value.

In a preferred embodiment, the assay involves binding the polypeptide using a binding agent immobilized on a solid support and isolating the polypeptide from the remainder of the sample. The bound polypeptide is then detected using a detection reagent containing a reporter group that specifically binds to the binding agent/polypeptide complex. These detection reagents may contain, for example, a binding agent that specifically binds to the polypeptide, or an antibody or other agent that specifically binds to the binding agent, such as anti-immunoglobulin, protein G, protein A, or lectins. Alternatively, competition assays can be used in which the polypeptide is labeled with a reporter group and the polypeptide and the binding agent are allowed to bind after incubation of the immobilized binding agent with the sample. The degree to which components in the sample inhibit the binding of the labeled polypeptide to the binding agent is indicative of the reactivity of the sample with the immobilized binding agent. Polypeptides suitable for use in these assays include the full-length annexin A3 protein described above and portions thereof that bind the binding agent.

As noted above, it is also possible, or alternatively possible, to detect drug resistance based on the level of mRNA encoding the annexin A3 protein in a sample of cancer cells. For example, at least two oligonucleotide primers can be used to amplify a portion of tumor cDNA derived from a sample in a polymerase chain reaction based assay, wherein at least one pair of the oligonucleotide primers encodes the annexin A3 protein The polynucleotide is specific for (ie hybridizes to) it. The amplified cDNA is then separated and detected using techniques well known in the art, such as gel electrophoresis. Similarly, an oligonucleotide probe that specifically hybridizes to a polynucleotide encoding annexin A3 protein can be used in a hybridization assay to detect the presence or absence of a polynucleotide encoding the tumor protein in a biological sample.

The present invention also provides kits for any one of the above detection and diagnosis methods. These kits typically contain two or more components necessary to perform the diagnostic assay. These components can be compounds, reagents, containers and/or equipment. For example, a container of the kit may contain a monoclonal antibody or fragment thereof that specifically binds to an annexin A3 protein, or a container of the kit may contain a nucleic acid probe or primer specific to an annexin A3 coding sequence.

cancer treatment

In other aspects of the present invention, it relates to the treatment of cancer based on the correlation between annexin A3 and the resistance of cancer to platinum chemotherapeutic drugs discovered for the first time by the inventors. In these methods, the patient's drug resistance is typically identified by the diagnostic method of the present invention, and then the patient's platinum-based chemotherapy drug administration regimen is determined based on the drug resistance result. In one embodiment, the treatment method of the present invention comprises administering platinum-based chemotherapy drugs after reducing the drug resistance of patients determined to have drug resistance through the method of modulating drug resistance of the present invention. In another embodiment, the treatment method of the present invention includes adjusting the dosage and frequency of administration of platinum-based chemotherapy drugs according to the determined drug resistance results. In yet another embodiment, the method of treatment of the present invention includes substituting platinum-based chemotherapy drugs for the treatment of the patient based on the determined drug resistance results. "Patient" as used herein refers to any warm-blooded animal, such as a mammal, preferably a human.

pharmaceutical composition

In other embodiments, the invention involves one or more polynucleotides, polypeptides, antisense oligonucleotides, ribozymes, oligonucleotide aptamers and/or antibodies disclosed herein in a pharmaceutically acceptable carrier or A formulation in a vehicle for administration to a cell or animal, alone or in combination with one or more other forms of diagnostic/therapeutic methods. In one embodiment, the pharmaceutical composition of the present invention can be used for diagnosing the resistance of platinum chemotherapy drugs in patients of the present invention and predicting/prognosing the therapeutic effect of the chemotherapy drugs. In another embodiment, the pharmaceutical composition of the present invention can be used to adjust the drug resistance of platinum chemotherapy drugs in the patient of the present invention. In this case, preferably, the pharmaceutical composition of the present invention can target the tumor cells of the patient and cause The way of changing (preferably down-regulating) the expression of annexin A3 is to adjust the drug resistance of patients.

The formulation of pharmaceutically acceptable excipients and carrier solutions is well known to those skilled in the art. Likewise, the development of suitable dosing and treatment regimens for use in various therapeutic regimens of the particular compositions described herein (including, for example, oral, parenteral, intravenous, intranasal, and intramuscular administration and pharmaceutical formulations) are also well known to those skilled in the art.

The route of administration and frequency and dosage of the therapeutic compositions described herein will vary from individual to individual and can be determined by the clinician. In general, these pharmaceutical compositions can be administered by injection (eg intradermal, intramuscular, intravenous or subcutaneous), nasal route (eg by inhalation) or orally. In general, appropriate dosages and treatment regimens provide an amount of active ingredient sufficient to obtain therapeutic benefit, which can be monitored or determined by determining improved clinical outcomes in treated patients compared to untreated patients.

The present invention is further illustrated by the following examples. However, it should be understood that these examples do not limit the scope of the present invention in any way.

Example

In the study of the present application, the inventors of the present application used proteomics technology for the first time to study the differences in protein expression profiles between drug-resistant and sensitive cells, and then searched for possible drug resistance markers.

Firstly, the ovarian cancer cell line SKOV3 resistant to cisplatin induced by the high-dose pulse method and the low-dose intermittent method was established. Through the detection of the biological characteristics of the two cell lines, it was found that different induction methods of the same drug have different effects on resistance to cisplatin. The expression of drug genes has different effects, and the small-dose intermittent method is more likely to produce drug resistance. And it has established a good cell model for studying the mechanism of platinum resistance.

Secondly, two ovarian cancer sensitive cell lines, SKOV3 and A2780, and four drug-resistant cell lines, SKOV3/CDDP, SKOV3/CBP, A2780/CDDP, and A2780/CBP, were used as research objects to establish their respective protein expressions by two-dimensional gel electrophoresis. Spectra, statistical analysis of differentially expressed proteins, mass spectrometric identification. The proteins annexin A3, destrin, IDHc, GSTO1-1 and cofilin 1 were differentially expressed in more than three drug-resistant cells. Among them, it was found for the first time that the expression of annexin A3 was significantly up-regulated in drug-resistant cells, and this result has been verified by RT-PCR and western blot.

In order to understand the relationship between annexin A3 and drug resistance, using genetic engineering technology, using pcDNA3.1/myc-His(-)B as a vector, constructing a eukaryotic expression plasmid expressing antisense annexin A3, using pcDNA3.1(+) As a vector, construct a eukaryotic expression plasmid expressing the sense annexin A3, and use pEGFP-N1 as a vector to construct a eukaryotic expression plasmid expressing annexin A3 fusion protein, and transfect each cell line through liposome-mediated plasmid DNA. SKOV3 and A2780 cell lines were transfected by pEGFP-N1-annexin A3, and the transfection efficiencies of SKOV3 and A2780 were 20-25% and 45-55%, respectively. After stable transfection, the drug resistance index of sensitive cell lines increased by 2 to 4 times after transfection of the positive-sense annexin A3 plasmid, and the drug resistance index of drug-resistant cell lines decreased by about 2 times after transfection of the antisense annexin A3 plasmid, which proved the up-regulation of annexinA3 Or downregulation is associated with drug resistance.

In order to clinically verify the correlation between annexin A3 and platinum-based drug resistance, surgical cases of platinum-based chemotherapy-sensitive and drug-resistant patients were collected respectively, and annexin A3 immunohistochemical test was performed on paraffin sections. The slides were read and evaluated under a double-blind method, and the rank sum test of graded data was used to count the expression levels of annexin A3 in chemotherapy-sensitive and drug-resistant patients. U value = 139, P value = 0.035<0.05, indicating that chemotherapy-sensitive patients and drug-resistant patients There was a significant difference in the expression of annexin A3 in chemotherapy-resistant patients, and the expression of annexin A3 in chemotherapy-resistant patients was higher than that in sensitive patients, which verifies that annexin A3 is a platinum resistance-related protein in ovarian cancer.

Example 1 Establishment and identification of epithelial ovarian cancer drug-resistant cell lines

(1) Experimental method

1) Cell lines and cell culture

The human serous ovarian cancer SKOV3 cell line was purchased from the Basic Medical Cell Center of the Chinese Academy of Medical Sciences. The cells were grown adherently in DMEM (HG) (Gibco, USA) medium containing 10% fetal bovine serum, cultured in a 37°C, 5% CO ₂ saturated humidity incubator, and digested and passaged with 0.25% trypsin.

2) Establishment of drug-resistant cell lines

1. The high-dose shock induction method is named as SKOV3/CDDP-P cells. After adding 100 μmol/L CDDP (cisplatin, FH Keding Co., Ltd.) to the medium for 2 hours, discard the drug-containing medium, add the drug-free medium, and place it in a 37°C, 5% _CO2 saturated humidity incubator to continue After culturing, the cells grow to the logarithmic growth phase (the density reaches 70%-80%), and the dose is repeatedly impacted. 100μmol/L impact 20 times, 200μmol/L impact 10 times. Dosing intervals gradually changed from the initial 4 weeks to 4-5 days. The specific administration time and interval are shown in Figure 1.

2. The small-dose intermittent induction method is named SKOV3/CDDP-80. Add 10 μmol/L CDDP to the culture medium, place in a 37°C, 5% CO ₂ saturated humidity incubator and culture for 48 hours, discard the drug-containing medium, add a drug-free medium, place at 37°C, 5% CO ₂ Continue culturing in a saturated humidity incubator until the cells grow to the logarithmic growth phase, and repeat the dose induction. The doses were 10, 20, 40 and 80 μmol/L each in 10 increments. The dosing interval was gradually changed from the initial 6 weeks to 1-2 weeks. The specific administration time and interval are shown in Figure 2.

After the cells were successfully induced, the cells were cultured in a drug-free medium for 2 months, and then various experiments were performed.

3) Drug sensitivity test

Cells in the exponential growth phase were taken and digested with trypsin to make a single-cell suspension. According to 2000 cells/well/100 μl, 6 parallel wells were set up for each concentration of each cell, and 12 wells were controlled. No drug was added, of which 6 3 wells were negative controls (adding cells without adding drugs), and 6 wells were blank controls (only adding culture medium). After 24 hours of cultivation at 37°C and 5% CO ₂ , add CDPP, dilute it into 7 gradients by 3-5 times, the highest concentration of CDDP is 200 μg/ml, continue to cultivate for 72 hours, add 5 mg/ml MTT (tetramethyl azozolium salt (Sigma, USA) 20 μl, 37°C, 5% CO ₂ Continue to cultivate for 4 hours, control the medium, add 100 μl lysate (containing 20% SDS, 50% NN dimethylformamide, pH 4.7), overnight, all The automatic microplate reader measures the absorbance value (OD value) at 540nm, and can calculate the average value of the absorbance value of each drug concentration, and calculate the cell survival rate and inhibition rate. According to the drug concentration and inhibition rate, use SPSS11.5 software to calculate IC50( Drug concentration that inhibits 50% cell growth) and drug resistance index (RI).

Inhibition rate = [(OD control - OD experiment)/OD control] × 100%

RI = IC50 of drug-resistant cell line/IC50 of sensitive cell line

4) Morphological observation

1. Inverted Microscope

Inoculate 5×10 ⁴ /ml single cell suspension into a 24-well culture plate lined with small glass slides, culture in a 37°C, 5% CO ₂ incubator for 72 hours, wash twice with PBS, and fix with formaldehyde for 10 minutes, Swiss-Giemsa staining for 2 hours, washed twice with PBS, dried at room temperature, sealed with neutral gum, observed and photographed under a light microscope. Swiss-Giemsa stain recipe:

Disodium hydrogen phosphate (1/15M) 6ml

Potassium dihydrogen phosphate (1/15M) 4ml

Wright's staining solution (Sigma) 120μl

MAY staining solution (Sigma) 120μl

Giemsa staining solution (Sigma) 500μl

2. Intracellular Ultrastructure Observation

(1) For cells growing in logarithmic phase (total number of cells ≥ 1×10 ⁶ ), discard the medium and wash twice with PBS;

(2) Add 10ml of PBS and send to the Electron Microscope Room of the Instrument Testing Center of the Academy of Military Medical Sciences (No. 27, Taiping Road, Haidian District, Beijing) for cell sectioning;

(3) Cells grow to the logarithmic growth phase, wash twice with PBS, scrape the cells with a cell brush, centrifuge at 1000g for 10 minutes, fix with 3% glutaraldehyde (1/15M PBS pH7.4), and fix at 4°C for 2 hours , rinse with 1/15M PBS+0.19M sucrose buffer for 15 minutes at 4°C. Gradient dehydration with ethanol at 4°C (50% ethanol, 70% ethanol, 90% ethanol, 90% ethanol+90% acetone, 90% acetone, 100% acetone for 10 minutes each), and 100% acetone for 10 minutes at room temperature. 100% acetone: embedding agent (1:1) was soaked at room temperature for 30 minutes, and pure embedding agent was soaked overnight. For embedding: the polymerization time is 35°C, 12 hours; 45°C, 12 hours; 60°C, 24 hours. ULTRACUTE/S microtome (RMC Company, USA) was used for ultrathin sectioning, uranyl acetate staining solution was used for 10 minutes in the dark, and lead citrate staining was performed for 10 minutes.

(4) Voltage 75kV, observed with EM400T electron microscope (Philips, Netherlands), and photographed with ×8000 and ×22000 electron microscopes.

5) Determination of cell population doubling time

Take the non-drug-resistant and drug-resistant cells in the logarithmic growth phase, inoculate 5000 cells per well in a 24-well culture plate, and culture at 37°C and 5% CO ₂ . The cells in 3 wells were taken every day for counting, and the observation was continued for 7 days. Plot the growth curve and calculate the population doubling time.

6) Cell cycle analysis

Take 1×10 ⁶ non-drug-resistant and drug-resistant cells in the logarithmic growth phase each to make a single-cell suspension, collect the cells by centrifugation, wash twice with cold PBS (pH 7.4), remove the supernatant, and add 70% cold Fix with ethanol and 3% serum in PBS (-20°C) overnight, centrifuge at 2000g to remove the supernatant, wash twice with cold PBS and resuspend with 0.5-1ml PBS (containing 200μg/ml RNase A), after mixing, 37 ℃ water bath for 30 minutes, add 400 μl propidium iodide (concentration 50 μg/ml, Sigma), 4 ℃ dark staining for 30 minutes, and measure the DNA content of 10000 cells with FACSCalibur (BD Company, USA) flow cytometer.

Seven) RT-PCR

1. Cell RNA extraction: Take about 5×10 ⁶ cells in the logarithmic growth phase, digest with 0.25% trypsin, pipette with PBS, elute into a 10ml centrifuge tube, centrifuge at 800rpm for 10 minutes, wash twice with PBS, and transfer the cells Transfer to a new Eppendorf tube, centrifuge, and discard the supernatant. Add 500 μl TRIzol (Sigma) to mix, pipette to mix, add 500 μl TRIzol, shake for 30 seconds, leave at room temperature for 5 minutes, add 200 μl chloroform, invert and mix for 15 seconds, and place at room temperature for 2 minutes, centrifuge at 12,000 g at 4°C After centrifugation for 15 minutes, take the upper aqueous phase and transfer it to a new Eppendorf tube. Precipitate RNA with 500 μl of isopropanol, mix well, and centrifuge at 12,000 g for 10 minutes at 4°C. Pour off the supernatant, wash the precipitate with 70% ethanol, and wash at 4°C. Centrifuge at 8000g for 10 minutes, suck off the supernatant, dry briefly for 15 minutes, and add 20 μl RNase-free water.

2. Reverse transcription (reverse transcription kit from Japan TaKaRa Company): In a 50 μl reaction volume, add 7 μl RNA, 10 mM dNTP 5 μl, 25 mM magnesium chloride 10 μl, 2.5 pmol/μl oligo dT 1 μl, 10×RT Buffer 5 μl, 40U/ml RNasin 0.5 μl, 5U/ml AMV 1 μl, RNase Free dH ₂ O 20.5 μl, mix well and centrifuge for a few seconds, 42°C water bath for 60 minutes, 98°C for 5 minutes to inactivate AMV.

3. PCR reaction, reaction system (total volume 20ul):

Taq MasterMix buffer (USA

10ul

Invitrogen)

Upstream primers 0.5ul

Downstream primers 0.5ul

Reverse transcription product 1ul

H ₂ O 8ul

The reaction conditions of each primer (Table 1) were pre-denatured at 95°C for 2 minutes and cycled, denatured at 95°C for 20 seconds, annealed at 58°C for 1 minute, extended at 72°C for 50 seconds, and extended at 72°C for 10 minutes after 35 cycles. 1.5% agarose to separate PCR products. After electrophoresis, put the agarose gel into the dark box of the JS-380 automatic gel image analyzer (Shanghai Peiqing Technology Co., Ltd.), and scan it with the gelscan system. Scan results were analyzed using QCapturePro software. β-actin was used as an internal reference for semi-quantitative comparison.

Eight) Western blot

1. Lysis of cells:

Take non-drug-resistant and drug-resistant cells in the logarithmic growth phase, trypsinize, centrifuge at 1500rpm for 5 minutes, discard the supernatant, add sterile PBS (pH7.4) to resuspend, perform cell counting, centrifuge at 1500rpm for 5 minutes, discard For the supernatant, wash the cells twice with cold PBS, and aspirate the supernatant. About 10 ⁶ cells were added with laemmli lysate (Bio-Rad, USA). When the cells were lysed, other cells were placed in an ice-water bath. After adding the lysate, boil at 99°C for 10 minutes, centrifuge at 10,000g at 4°C for 10 minutes, take it out and bathe in ice water. After taking out the sample for protein concentration determination, add 5 μl of 2-ME (2-mercaptoethanol) to the remaining sample per 100 μl, centrifuge at 10000 g, 4°C for 10 minutes, and store at -20°C.

2. Determination of protein concentration by BCA method

Take the BCA Protein Quantification Kit (Pierce) A solution and B solution and mix it at 50:1 (v:v), take the above sample and add it to 500 μl AB mixed solution, mix well, add 2 μl lysate solution to 500 μl AB mixed solution for the blank control , in a water bath at 37°C for 30 minutes, zeroed with a blank control, and measured the OD value at a wavelength of 562nm on a Smartspec ^TM plus spectrophotometer (Bio-Rad). Use Excel to draw a standard curve and calculate the sample concentration.

3. SDS-PAGE electrophoresis

Prepare Tris-glycine electrophoresis buffer: 25mmol/L Tris, 250mmol/L glycine, 0.1% SDS. Prepare 5ml of 10% separating gel: 1.9ml of deionized water, 1.7ml of 30% acrylamide solution, 1.3ml of 1.5% Tris (pH8.8), 0.05ml of 10% SDS, 0.05ml of 10% ammonium persulfate, and 0.002ml of TEMED. The above glue solidified after 30 minutes. Prepare 2ml of 5% stacking gel: 1.4ml of deionized water, 0.33ml of 30% acrylamide solution, 0.25ml of 1.0% Tris (pH 6.8), 0.02ml of 10% SDS, 0.02ml of 10% ammonium persulfate, and 0.002ml of TEMED. The above glue solidified after 30 minutes. Take 30 μg of protein from each cell and load it at 80-100v for 2 hours of electrophoresis.

4. Electrotransfer

Prepare transfer buffer: 48mM Tris HCl, 39mM glycine, 0.037% SDS, 20% methanol. Two sheets of filter paper and a PVDF membrane (Pierce) were cut to the size of the gel while wearing gloves. Soak the above-mentioned filter paper and filter membrane with transfer buffer for 10 minutes, and stack them according to filter paper, filter membrane, gel, and filter paper, and pay attention to avoid air bubbles. bottom right corner mark. 80v, ice-water bath electrotransfer for 2 hours.

5. Western Blotting

Prepare 10×TBST: 200mM Tris HCl, 1.5mM NaCl, add 0.05% Tween-20 before use. Remove the PVDF membrane, shake and wash with TBST for 5 minutes, and block in TBST containing 5% skimmed milk powder for 1 hour. Shake and wash three times with TBST (15 minutes), add the primary antibody prepared in blocking solution, and shake gently overnight at 4°C. Shake and wash three times with TBST (15 minutes), add secondary antibody (HRP-labeled) prepared in blocking solution, and shake gently for 1 hour. Shake three times with TBST. Mix the chemiluminescent substrate solution ECL (Pierce) A and B by volume 1:1, apply the PVDF membrane for 5 minutes, and expose to the dark room for color development.

9) Statistical analysis

The experimental data were processed using SPSS11.5 statistical software, and statistical analysis was carried out by t test.

(2) Experimental results

1) Drug resistance index

The IC50 of SKOV3 against CDDP was (6.67±2.58) μmol/L, and the RI was 1.

SKOV3 cells were induced in vitro by high-dose shock induction method and small-dose intermittent induction method. It took 16 months to obtain two drug-resistant cell lines, and the drug-resistant cell lines were stable for 4 months in drug-free medium. The IC50 of SKOV3/CDDP-P on CDDP was (27.24±18.60) μmol/L, and the RI was 4.12±2.01; the IC50 of SKOV3/CDDP-80 on CDDP was (76.07±3.85) μmol/L, and the RI was 11.50±3.15. The drug resistance index of the low-dose intermittent induction method was higher than that of the high-dose pulse induction method (P<0.05) (Figure 3), and the low-dose intermittent induction was more likely to produce drug resistance than the high-dose pulse induction.

2) Observation of cell morphology

1. Under a light microscope: SKOV3 cells and other induced drug-resistant cell lines grow adherently. Under a light microscope, SKOV3 cells grow in a spindle-shaped, epithelial-like manner with clear borders and good refraction (Fig. 4a). After drug induction, most of the cells died, and the shape of the cells became elongated, with fuzzy borders and different sizes. Among them, the changes of SKOV3/CDDP-80 cells were more obvious, a large part of the cells grew like neurons, protruding pseudopodia, more common nuclear abnormalities, and increased cytoplasmic granules (Fig. 4b,c). In the logarithmic growth phase, the cell morphology gradually recovered, and the morphology of SKOV3/CDDP-P cells was not significantly different from that of SKOV3 cells, while the SKOV3/CDDP-80 cells were still pleomorphic with unclear borders (Fig. 4d, e, f) .

2. Under the scanning electron microscope, the normal SKOV3 cells are spindle-shaped, with abundant, long and slender microvilli on the cell surface, evenly distributed, large and round nuclei, and bulge toward the cell surface. Both CDDP-P and CDDP-80 showed that the microvilli on the cell surface were sparse and unevenly distributed. CDDP-P had little morphological changes, but CDDP-80 had significant morphological changes, with cells extending from the nucleus to both sides with a larger area (Fig. 5a). Under the transmission electron microscope, it can be seen that the ratio of nucleus to plasma in SKOV3 cells is inverted, the nucleus is large, containing 1 to 2 nucleoli, the nuclear membrane is complete and smooth, the chromatin in the nucleus is aggregated into clumps, and the cytoplasm contains a large number of mitochondria. The nuclei of CDDP-P and CDDP-80 were large and irregular in shape, deformed nuclei were visible, the nuclear membrane was obviously depressed, and there were nuclear pockets and nuclear processes. The chromatin is unevenly distributed, some condense into blocks, and the mitochondria in the cytoplasm are reduced, with a large number of vesicle-like structures. These vesicle-like structures were partially vacuolated and partially granule-like (Fig. 5b, c).

3) Growth curve and cell population doubling time

On the second day, all the cells were in the residence period, and the number of cells did not increase significantly. From 2 to 3 days, SKOV3 cells entered the logarithmic growth phase, which was significantly higher than that of SKOV3/CDDP-P and SKOV3/CDDP-80 (Figure 6). The population doubling time (GDT) of SKOV3, SKOV3/CDDP-P and SKOV3/CDDP-80 were 27.49±4.21h, 47.26±4.64h and 57.48±6.17h, respectively. The GDT of SKOV3/CDDP-P and SKOV3/CDDP-80 cells was significantly higher than that of SKOV3 cells (P<0.01, P<0.001). However, SKOV3/CDDP-80 cells grew the slowest, which was significantly different from SKOV3/CDDP-P cells (P<0.01).

4) Cell cycle distribution

The G ₀ /G ₁ phase of SKOV3/CDDP-P and SKOV3/CDDP-80 were significantly prolonged (P<0.01), and the S phase and G ₂ /M phase were shortened (P<0.05, P<0.01). Compared with SKOV3/CDDP-P, SKOV3/CDDP-80 had no significant difference in each phase, but the G ₀ /G ₁ phase tended to be prolonged (Table 2).

5) Expression of drug resistance-related genes

RT-PCR method was used to detect multidrug resistance protein (MDR1), multidrug resistance associated protein 1 (MRP1), lung resistance protein 1 (LRP1) and glutathione Expression of glutathione S-transferase pi (glutathioneS-transferase pi, GST-pi) mRNA. SKOV3 does not express MDR1, but expresses MRP1, LRP1, and GST-pi. In SKOV3/CDDP-P, MDR1 was up-regulated (P<0.01), both MRP1 and LRP1 were down-regulated (P<0.01), and GST-pi had no significant change. MDR1 of SKOV3/CDDP-80 was up-regulated (P<0.01), while MRP1, LRP1, and GST-pi had no significant changes (Fig. 7, 8, 9). The different induction methods of the same drug have different effects on the expression of drug resistance genes, suggesting that the drug dose has different mechanisms for the generation of drug resistance, and it also shows that the drug dose has a certain impact on the generation of drug resistance cases.

(3) Conclusion

In this study, in order to clarify the differences between the drug-resistant cell lines established by different induction methods and provide a more ideal drug-resistant model for clinical research, the human ovarian serous papillary cystadenocarcinoma SKOV3 cell line was selected as the experimental object, and CDDP was used as the induction method. Drugs, two drug-resistant cell lines-SKOV3/CDDP-P and SKOV3/CDDP-80 were established by using the high-dose pulse method and the small-dose intermittent induction method for 16 months. It can be seen that SKOV3 is a slow process for developing drug resistance to CDDP. process. At the same time, the results of low-dose intermittent induction are more likely to produce drug resistance than high-dose pulse induction suggest that the dose of clinical chemotherapy drugs has a certain impact on the generation of drug-resistant cases. In the drug-resistant cell line established in this experiment, during the 4-month routine culture process, the drug-resistant indicators did not change, and the drug resistance was very stable. Different induction methods of the same drug have different effects on the expression of drug resistance genes, suggesting that drug doses have different mechanisms for the generation of drug resistance.

Example 2 Screening and Validation of Platinum Resistance-Related Proteins in Epithelial Ovarian Cancer

(1) Experimental method

1) Cells and cell culture: human serous ovarian cancer cell line SKOV3 was purchased from the Basic Medical Cell Center of the Chinese Academy of Medical Sciences. The SKOV3/CDDP line was induced into drug-resistant cell lines by high-dose cisplatin pulse method in our laboratory (see the first part for the specific induction method. Since the high-dose pulse induction method is similar to the clinical chemotherapy method, SKOV3/CDDP was used in subsequent studies. -P is the research object and named as SKOV3/CDDP). Human epithelial ovarian cancer cell lines A2780, A2780/CDDP, A2780/CBP and SKOV3/CBP were provided by Professor Li Li (Department of Gynecologic Oncology, Cancer Hospital Affiliated to Guangxi Medical University). All cells were grown adherently in DMEM (HG) medium containing 10% fetal bovine serum, and cultured in a 37°C, 5% CO ₂ saturated humidity incubator.

2) Drug susceptibility test: see the first part for specific experimental procedures.

3) Two-dimensional gel electrophoresis

1. Preparation of total cell protein: After the cells are cultured to the logarithmic growth phase, digest with trypsin, centrifuge in a centrifuge tube, wash twice with cold PBS, centrifuge to remove the supernatant, collect cells 1×10 ⁶ and suspend in 50ul lysate (2M thiourea, EDTA 1mM, 7mol/L urea, 4% CHAPS, 40mmol/L Tris, 65mmol/L DTT), pipette to mix, shake for 15 seconds, fast freeze-thaw for 3 cycles, sonicate until transparent, add appropriate amount Cocktail protease inhibitor (Roche, Germany), 20ug/ml DNase I, 5ug/ml RNaseA (Sigma), ice bath for 30 minutes, centrifuge at 13000g, 4°C for 30 minutes, take the supernatant into another Ep tube, and use the Bradford method (Bio-Rad) was used to determine the concentration of protein, and the samples were divided and stored at -70°C for later use.

2. The first phase solid-phase pH gradient isoelectric focusing electrophoresis: total cell protein extract (1mg) and hydration solution (8mol/L urea+4%CHAPS+20mmol/L DTT+0.5%IPG bufferpH3-10NL+trace bromine Phenol blue) mixed thoroughly, with a total volume of 350ul, added to the IPGstrip holding tank, and the IPG dry strip pH3-10 (180mm×3mm×0.5mm) (Amersham Biosciences, UK) with the protective glue side down, gently put it into the holder In the glue tank, remove air bubbles, cover with a layer of DryStrip Cover Fluid (Amersham Pharmacia, #17-1335-01), cover the lid of the glue tank, place on the electrode plate of IPGphor isoelectric focusing instrument (Amersham Biosciences), hydrate and Focusing is carried out automatically at 20°C, with a total voltage-time product of 60,000-80,000 Vh, including hydration re-foaming for 4 hours, 6 hours at a low voltage of 30V, and then 500V1h, 1000V1.5h, 5000V1h, and finally stabilized at 8000V.

3. Equilibration: After the isoelectric focusing is completed, quickly take out the IPG strips with samples and place them in 20ml of equilibrium solution A (50mmol/L Tris-HCl pH=8.8+6mol/L urea+30%glycerol+2%SDS+0.3% DTT) and 20ml of equilibrium solution B (50mmol/L Tris-HClpH=8.8+6mol/L urea+30% glycerol+2%SDS+1.85% iodoacetamide+trace bromophenol blue) each equilibrated for 15 minutes.

4. The second phase of vertical plate SDS-PAGE electrophoresis: move the equilibrated IPG gel strip to the upper end of the stacking gel of a 1.5mm thick continuous gel (13% homogeneous separation gel), and add SDS standard protein on the outside of one end , seal with 0.5% agarose after removing air bubbles, cool with 15C circulating water, first use 40mA/2 gels for constant current electrophoresis for 40 minutes, then use 60mA/2 gels for constant current electrophoresis until bromophenol blue reaches the lower edge of the glass plate Stop electrophoresis.

4) Coomassie brilliant blue staining:

1. Take out the gel plate, place it in fixative solution (40% ethanol, 10% glacial acetic acid, 50% double distilled water), and shake for 30 minutes.

2. Discard the fixative, and place it in the staining solution (a piece of R-350 (Amersham Pharmacia) dissolved in 1600mL of 10% acetic acid) for heat staining for 10 minutes.

3. Place in decolorization solution (10% glacial acetic acid) and shake overnight.

5) Gel image analysis: The colored gel was imaged by an Imagescanner scanner (Amersham Biosciences) and a Labscan5 scanning software (Amersham Biosciences). The amount of protein is defined as the sum of all pixel values that make up this blob. In order to more accurately reflect the changes in the amount of protein spots, the content of each point is expressed as a relative percentage (%Vol), that is, the pixel value of a protein spot accounts for the percentage of all protein spot pixel values in the entire gel. ImageMaster2D Platinum analysis software (Amersham Biosciences) was used to analyze the image for background reduction, spot detection, matching, and quantitative acquisition of spot position coordinates. All data analyzes were performed on SPSS 11.5 software.

6) Preparation of mass spectrometry samples:

1. Decolorization

Use a clean scalpel to cut off the protein bands in the gel after electrophoresis and staining, and cut the tape into 1-2 mm ³ gel particles and place them in Eppendorf tubes. After adding ultrapure water to the Eppendorf tube to wash several times, soak the colloid with 30 μl of 50 mM ammonium bicarbonate (NH ₄ HCO ₃ ) decolorization solution containing 50% acetonitrile (ACN). Shake for 20 minutes, discard the solution, and repeat 2-3 times until the blue color in the gel fades away.

2. Enzyme hydrolysis

The decolorized colloidal particles were vacuum centrifuged and dried for 30 minutes to make them completely dehydrated, and the volume was reduced. Add 3-10 μL of trypsin solution (0.01 μg/μl) and place in a refrigerator at 4°C for 20-30 minutes. Take out the sample from the refrigerator and carefully observe the amount of enzyme in the sample. If the enzyme solution is still left after the gel particles are completely swollen, suck out the excess enzyme solution and add 5-10μl 25mM NH ₄ HCO ₃ solution as a buffer for the incubation reaction . Seal the sample tube with Parafilm, sonicate for 1-2 minutes, and keep it in a 37°C water bath overnight.

3. Peptide Extraction

The sample in step 2 was taken out, and the supernatant was aspirated by centrifugation and placed in a new Eppendorf tube (2 μl of the supernatant can be taken out directly for mass-assisted laser desorption ion time-of-flight mass spectrometry (MALDI-TOF-MS)).

Add about 5-100 μl of 5% TFA to the gel, just to cover the gel particles. Seal the mouth of the tube with a parafilm and sonicate for 1-2 minutes, incubate in a water bath at 37°C for 1 hour, and suck out the supernatant after centrifugation. Be careful not to suck the colloidal particles into the pipette tip or bring them out to cause sample loss.

Add 5% trifluoroacetic acid (TFA) : acetonitrile 1:1 mixture 5-100μl to the gel, seal the tube opening with a parafilm and sonicate for 1-2 minutes, incubate in a water bath at 37°C for 1 hour, centrifuge and aspirate Serum.

Add an appropriate amount of ACN and let it stand at room temperature for a while, until the colloidal particles are dehydrated and turn white, absorb and combine the supernatant.

4. Dry

The supernatant obtained from the above 4 steps of extraction was freeze-dried.

7) Identification of protein by peptide mass fingerprinting: add 2 μl-7 μl 0.5% TFA to the dried peptide mixture, after mixing, take 0.5 μl-1 μl solution and an equal volume of saturated matrix solution (alpha-cyano-4-hydroxyl Cinnamic acid (Germany Bruker Company) was dissolved in 50% ACN solution containing 0.1% TFA) and mixed, then added to the stainless steel target, dried at room temperature, and then installed on the target for measurement. Data were acquired on a Bruker reflex III MALDI-TOF-MS mass spectrometer (Bruker) at 20 kV in positive ion mode. The mass accuracy of the mass spectrum peaks was calibrated using the self-cleaved peak of trypsin as an internal standard.

Eight) Database query:

The SwissProt and NCBInr databases were searched by Mascot software. Query conditions: The quality of the peptide fragments in the peptide quality fingerprint is not limited, trypsin is selected as the enzyme, carbamidomethylation and oxidation are selected as the variable modification, the maximum allowable error of the molecular weight of the peptide fragments is controlled within ±100ppm, and each peptide is allowed to have 1 For incomplete cleavage sites, humans are selected as the source of species, cations and monoisotopes are selected for ions, and the signals of the selected fragments should be stronger, more than 1 times the width of the baseline.

Nine) Quantitative PCR:

Cells in the logarithmic growth phase were collected, total cellular RNA was extracted, cDNA was synthesized by reverse transcription, and quantitative PCR reaction was performed. Primers were synthesized by Invitrogen (see Table 3). The optimized fluorescent real-time quantitative PCR system was 10 μl, including 1 μl primers, 1 μl template cDNA, 5 μl DyNAmo PCR Master Mix (Finnzymes, Finland), and 3 μl RNase-free water. The PCR conditions were set as follows: inactivation at 95°C for 2 minutes, denaturation at 95°C for 30s, annealing at 52°C for 50s, extension at 72°C for 40s, a total of 35 cycles, and a final extension at 72°C for 7 minutes after the cycle ended. Use Opticon Monitor 3 software (Bio-Rad) to analyze the CT (Threshold cycle) value of each test sample in the PCR process. This experiment uses a relative quantitative method and uses the ΔΔC _T method for calculation and comparison. Each experiment was repeated 3 times, and the mean value was taken.

10) Western blot: See the first part for specific experimental procedures.

11) Statistical methods

The differentially expressed protein spots in samples were detected by one-way analysis of variance and t test, and the statistical analysis was performed in SPSS 11.5.

(2) Experimental results

1) Drug-resistant cell lines

The IC50 and RI of the six kinds of cells to cisplatin or carboplatin are shown in Table 4. The drug-resistant properties of the four kinds of drug-resistant cells are relatively stable, and there is no significant change in the detected RI during the culture in conventional medium. Therefore, our experimental objects are stable, increasing the reliability and repeatability of experimental results.

2) Two-dimensional gel electrophoresis patterns of human ovarian cancer cell lines and their drug-resistant cell lines

Two-dimensional gel electrophoresis was used to separate the protein samples of each type of cell, and at least 3 gels were repeated for each cell sample, and about 1,800 protein spots could be detected in each gel after staining. The gel matching rate of a cell sample can reach more than 95%, the gel matching rate of a cell line and its sublines can reach 90%, and the gel matching rate of different cell lines can reach 80% to 85%. . The relative volumes of the protein spots corresponding to each other in all repeated gels of each drug-resistant cell sample were averaged, and the difference in expression level was considered as a difference point by more than 2 times. Comparing resistance to its sensitive counterpart cell samples identified differential protein spots associated with platinum resistance in different cell lines. These points are mainly distributed in the range of isoelectric point 5-9 and molecular weight 14.0kDa-70.0kDa.

3) Identification of proteins by peptide mass fingerprinting

Through software analysis, 62 differentially expressed protein spots (P<0.05) between human ovarian cancer drug-resistant cell lines SKOV3/CDDP, SKOV3/CBP, A2780/CDDP and A2780/CBP and their parental cells were obtained (Figure 10). MALDI-TOF-MS successfully identified 57 protein spots. In order to ensure the accuracy of the matching results, the matched protein spots on different gels were identified by mass spectrometry at the same time, and the results showed that they were the same protein. In the protein expression profiles of the four drug-resistant cells, we found that each drug-resistant cell had 12 to 16 differentially expressed proteins, a total of 36 proteins, and more than 1/3 of the differentially expressed proteins belonged to enzymes. Its functions include many aspects of metabolism, such as energy metabolism, nucleotide metabolism, redox, cytoskeleton-related proteins, protein and fat metabolism, signal transduction and so on.

4) Differentially expressed proteins between SKOV3/CDDP and SKOV3 cells

In the drug-resistant subline SKOV3/CDDP of SKOV3, 14 protein spots were found to be differentially expressed (P<0.05). Among them, the expression of 9 protein spots was up-regulated, and the expression of 5 protein spots was down-regulated (Table 5). Among these differentially expressed protein spots, some proteins such as the heat shock protein family (points 21 and 28) have been confirmed to be related to drug resistance, and their expression was up-regulated by 7 times in drug-resistant samples. The expression of Annexin A3 (point 25) increased 4-fold, while the expression of villin 2 (point 1) and IDHc (point 7) decreased 7-fold and 9-fold, respectively, compared with the parental cells.

5) Differentially expressed proteins between SKOV3/CBP and SKOV3 cells

Compared with SKOV3, SKOV3/CBP had a total of 16 protein spots differentially expressed (P<0.05) (Table 6). In SKOV3/CBP, some proteins showed different expression differences from SKOV3/CDDP. Stomatin (EPB72)-like 2 (point 4) was down-regulated in SKOV3/CDDP, but highly expressed in SKOV3/CBP. Heat shock 70kDa protein 1A and heat shock protein 27 also showed the opposite expression trend to SKOV3/CDDP. Expression of stathmin 1 (spot 18) and phosphoglycerate mutase 1 (spot 14) was upregulated four-fold and IDHc (spot 7) was downregulated three-fold. Annexin A3 (spot 25) was only upregulated 3-fold compared to SKOV3/CDDP. In previous studies, upregulation of annexin A1 (point 9) was associated with drug resistance, whereas annexin A1 was downregulated in SKOV3/CBP. Among the 16 differentially expressed proteins, the intensity of protein spots changed between 2 and 4 times.

6) Differentially expressed proteins between A2780/CDDP and A2780 cells

Since both A2780 and SKOV3 belong to epithelial ovarian cancer, the protein expression profile of A2780 cells is similar to that of SKOV3. In A2780-resistant cisplatin cells A2780/CDDP, there were 15 differentially expressed protein spots (P<0.05) (Table 7). Among them, only 4 protein spots were underexpressed. In A2780, the expression of annexin A3 (point 25) was underexpressed, while the expression of annexin A3 was increased by 20 times in A2780/CDDP. GSTO1-1 (point 54) and splicing factor, arginine\/serine-rich 3 (point 33) were up-regulated 5-fold and 6-fold, respectively.

Seven) Differentially expressed proteins between A2780/CBP and A2780 cells

Although both A2780/CBP and SKOV3/CBP were established under the induction of carboplatin, they differed in the expression of differential proteins. In A2780/CBP, 12 protein spots were found to be differentially expressed (P<0.05) (Table 8), among which 5 protein spots were down-regulated, and all of them were down-regulated by 2-3 times. Phosphoribosyltransferase (spot 32) and UMP-CMPK (spot 31) were up-regulated 3-fold and 4-fold, respectively. In SKOV3/CBP, ECH1 protein (spot 47) and stathmin 1 (spot 18) were slightly up-regulated, while in A2780/CBP down. The expression of Cofilin 1 (point 34) also showed the opposite trend, which was significantly upregulated (10-fold) in A2780/CBP, but downregulated in SKOV3/CBP. However, the expression of dUTP pyrophosphatase (point 56) all decreased.

Among the differentially expressed proteins of the four drug-resistant cells, we found that 5 proteins were differentially expressed in more than three drug-resistant cells (Table 9). Among them, the expressions of annexin A3 (Fig. 11, 12) and destrin were both up-regulated in drug-resistant samples, and annexin A3 was most significantly up-regulated (3-20 times) (Fig. 13). NADP-dependent isocitratedehydrogenase 1 (IDHc) was up-regulated in the four Both were down-regulated in drug-resistant cells (Figure 14). Glutathione transferase omega 1 (GSTO1-1) was upregulated in SKOV3/CDDP, A2780/CDDP and A2780/CBP, except that its expression was not changed in SKOV3/CBP (Figure 15). However, cofilin 1 showed completely opposite properties. The expression of cofilin 1 was down-regulated in the two drug-resistant cells of SKOV3, but it was up-regulated in the drug-resistant cells of A2780 (Figure 16).

Eight) Quantitative PCR method to detect the expression of mRNA

In order to verify the expression of the five proteins in SKOV3 and A2780 cells and their respective drug-resistant sublines, detection was performed at the RNA level. The results ( FIG. 17 ) showed that both SKOV3 and A2780 cells had low expression of annexin A3, while the levels of annexin A3 in their cisplatin- and carboplatin-resistant cells were significantly up-regulated (P<0.01). Destrin was up-regulated in SKOV3/CDDP, SKOV3/CBP and A2780/CDDP cells (P<0.01, P<0.05). Cofilin1 was only up-regulated in SKOV3/CBP (P<0.05), and down-regulated in A2780/CDDP (P<0.01). However, GSTO1-1 was only up-regulated in cisplatin-induced drug-resistant cells (P<0.01). Except for A2780/CDDP, IDHc was down-regulated in the other three drug-resistant cells (P<0.01, P<0.05).

9) Western blot to detect the expression of annexin A3

The four differentially expressed proteins annexin A3, destrin, cofilin 1 and GSTO1-1 were further verified by western blot (IDHc antibody was not purchased). As a result, it can be seen that the results of western blot (Figure 18) are completely consistent with the results of two-dimensional gel electrophoresis (2-DE). It is suggested that these five proteins play a certain role in the formation of drug resistance, and the up-regulation or down-regulation of these proteins may occur at different levels (RNA level or protein level or both) in drug-resistant cells.

(3) Conclusion

In our study, the drug resistance index of the four drug-resistant cells did not change significantly during the culture process. Therefore, our experimental object is stable, which increases the reliability and repeatability of the experimental results, and establishes a good cell model for the study of the mechanism of platinum resistance. In the protein expression profiles of the four drug-resistant cells, each drug-resistant cell had 12 to 16 differentially expressed proteins, a total of 36 proteins, and more than 1/3 of the differentially expressed proteins belonged to enzymes. Its functions include many aspects of metabolism, such as energy metabolism, nucleotide metabolism, redox, cytoskeleton-related proteins, protein and fat metabolism, signal transduction and so on. Among these differentially expressed proteins, we found that 5 proteins had significant changes in more than three samples, namely annexin A3, destrin, IDHc, GSTO1-1 and cofilin 1, among which annexin A3 had the most significant change (3～ 20 times), suggesting that these five proteins play a role in the formation of drug resistance, and the up-regulation or down-regulation of these proteins in drug-resistant cells may occur at different levels (RNA level or protein level or both Of).

Example 3 Functional identification of candidate drug resistance-associated protein annexin A3

Annexin A3 has been studied on platinum drug resistance. Since the results of quantitative PCR and western blot of annexin A3 are consistent with the results of 2-DE, and the most obvious up-regulation trend (3-20 times), annexin A3 was selected as the function Drug resistance-associated candidate proteins studied.

(1) Experimental method

1) Plasmid construction

1. Construct plasmids (pcDB-sense anx3 and pcDB-antisense anx3) containing the sense and antisense sequences of the annexin A3 (anx 3) target gene

(1) Primer design: Design full-length primers for the translation region of mRNA based on the human anx 3 sequence (gi: 4826642) known in Genbank. Sense 5′-gaattcCATCATGGCATCTATCTGGGTT-3′, Antisense 5′-gaattcGTCATCTCCACCACAGA-3′, the length of the PCR amplification product is 984bp, the italicized restriction site is added, and the restriction site EcoR I is added on both sides to synthesize T7 primer TAATACGACTCACTATAGGG, Primers were synthesized by Invitrogen.

(2) See the first part for the specific steps of extracting total RNA and reverse transcription

(3) PCR amplification: using cDNA as a template, the reaction volume is 25 μl, and the components are as follows: Pfu 10×Rxn buffer (Promega, USA) 2.5 μl, 10mM dNTP 2 μl, deionized water 19 μl, anx3 upstream and downstream primers (see the primer design section) ), 0.5 μl of each, 0.3 μl of pUC-anx3 plasmid, and 0.2 μl of Pfu DNA polymerase (Promega), placed in a PCR machine, denatured at 95°C for 1.5 minutes, and entered the cycle, denatured at 95°C for 1 minute, annealed at 50°C for 1 minute, and extended at 72°C for 1.5 min, 72°C extension for 10 min after 35 cycles.

Prepare electrophoresis solution: 0.04mol/L Tris-acetic acid, 0.001mol/L EDTA; prepare 1.0% agarose electrophoresis gel containing 0.5ug/ml EB, take 25μl of the reaction product into the well of the comb, and electrophoresis at 120V for 20 After electrophoresis, put the gel into the JS-380 automatic gel image analyzer and take pictures.

(4) DNA gel recovery: according to the instructions of the agarose gel DNA recovery kit of Tianwei Times Technology Co., Ltd.

1) Cut a single DNA band from the agarose gel with a clean blade, cut off as much excess gel as possible, and weigh it.

2) Add 3 times the volume of solution PN.

3) Water bath at 50°C for 10 minutes, during which time the centrifuge tube was continuously turned until the gel was completely dissolved.

4) Add the solution obtained in the previous step into an adsorption column, centrifuge at 13,000 rpm for 60 s, and discard the waste liquid in the collection tube.

5) Add 800 μl PW of washing solution, 13000 rpm, 60s, discard the waste solution.

6) Add 500 μl PW of washing solution, 13000 rpm, 60s, discard the waste solution.

7) Put the centrifugal adsorption column back into the collection tube, centrifuge at 13,000 rpm for 2 minutes, and remove the rinsing solution as much as possible.

8) Take out the adsorption column and put it into a clean centrifuge tube, add 20 μl membrane washing buffer EB in the middle of the adsorption membrane, place it at room temperature for 2 minutes, centrifuge at 13000 rpm for 60 seconds, and add the centrifuged solution back into the centrifugal adsorption column, 13000 Spin centrifuge for 60s.

Concentration measurement: Take 1 μl of the above solution into 99 μl of deionized water, adjust to zero with 100 μl of deionized water, use a UV spectrophotometer to measure the OD value at 260 nm, and calculate as 10D=50 μg/ml DNA. The ratio of OD260/OD280 should be between 1.7-1.9.

(5) Tailing reaction: carried out according to the method instructions attached to the pGEM-T vector of Promega Company. Reaction system 25 μl: Taq 10×buffer 2.5 μl, 10 mM dNTP 5 μl, 25 mM Mgcl ₂ 5 μl, Taq DNA polymerase (TaKaRa) 0.5 μl, PCR product 12 μl, placed in a PCR machine, and reacted at 72°C for 30 minutes.

(6) Solution recovery

The reaction solution was recovered with DNA Fragment Purification Kit Ver.2.0 (TaKaRa).

1) Add 100μl DB Buffer to the above reaction solution, and mix evenly.

2) Place the Spin Column in the kit on the Collection Tube.

3) Transfer the solution from the above operation 1 to the Spin Column, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid in the collection tube.

4) Add 500μl Rinse A to the Spin Column, centrifuge at 12000 rpm for 30s, and discard the waste liquid.

5) Add 700μl Rinse B to the Spin Column, centrifuge at 12000 rpm for 30s, and discard the waste liquid.

6) Repeat step 5.

7) Put the Spin Column into a clean 1.5ml centrifuge tube, add 20μl Elution Buffer in the middle of the SpinColumn membrane, place it at room temperature for 1min, and centrifuge at 12000rpm for 1min to elute the DNA.

(7) Ligation reaction with T carrier: Reaction system 10 μl: 10×T ₄ DNA Ligasebuffer 1 μl, T ₄ DNA Ligase (TaKaRa) 1 μl, deionized water 2 μl, A-tailed PCR product 5 μl, pGEM-T vector (Promega ) 1 μl, mix well and place overnight at 4°C.

(8) Preparation of Escherichia coli Competent Cells

1) Take out 100 μl of the original bacterial solution (stored at -80°C) and add it to 5ml LB bacterial culture medium, and culture overnight at 37°C with shaking at 200 rpm/min.

2) Take 50 μl of the bacteria solution from the previous step and transfer it to a centrifuge tube containing 5 ml of LB culture medium, and culture at 37° C. with shaking at 220 rpm/min for 3 hours. Monitor the growth of the culture by measuring the OD600 value every 20-30 minutes so that the number of viable cells should not exceed 10 ⁸ /ml.

3) Transfer the culture to a sterile, ice-precooled centrifuge tube and place on ice for 10 minutes.

4) Centrifuge at 4°C, 4000 rpm for 10 minutes, and discard the supernatant.

5) Resuspend the bacteria with 600 μl of pre-cooled 0.1 M calcium chloride and place in an ice bath for 30 minutes.

6) Centrifuge at 4000 rpm for 10 minutes at 4°C and discard the supernatant.

7) Resuspend bacteria with 2ml of pre-cooled 0.1M calcium chloride for every 50ml of initial culture, then aliquot into 50μl portions, store at 4°C for 12-24 hours for use. It can also be stored at -80°C for a long time.

(9) Transform competent cells:

1) Freshly prepared 200 μl competent cells were placed on ice, and after the ice was completely thawed, the cells were gently suspended evenly.

2) Add 10 μl of plasmid DNA and mix gently.

3) Place on ice for 30 minutes.

4) Heat shock in a water bath at 42°C for 90 seconds.

5) Place on ice for 2 minutes.

6) Add 150 μl of LB culture solution, shake at 100 rpm/min at 37°C for 45 minutes to recover.

7) Transfer 150 μl of transformed competent cells to the agarose medium containing antibiotics (Amp ⁺ ), add 20 μl of x-gal (20 mg/ml) and 4 μl of isopropyl-β-D galactose before coating solution (200mg/ml). To coat evenly, leave the plate at room temperature until the liquid is absorbed.

8) Invert the plate and incubate at 37°C for 12-16 hours.

After the plate was cultured overnight at 37°C, several blue and white spots were visible, and the white spots were picked and added to 5ml LB containing 90μg/ml Amp, 200rpm/min, 37°C overnight.

(10) Extraction of plasmid DNA (for specific steps, please refer to the instruction manual of the plasmid DNA extraction kit of Tianwei Technology Co., Ltd.)

1) Centrifuge 3ml of overnight cultured bacteria at high speed for 1 minute (4°C, 4000rpm, 10min), and completely remove the supernatant.

2) Add 200 μl of solution P1, and fully suspend the bacteria with a pipette tip or a shaker.

3) Add 200 μl of solution P2, gently invert 6-8 times to lyse the bacteria, and leave at room temperature for less than 5 minutes until the solution becomes clear.

4) Add 200 μl of solution PIII, gently invert up and down 6-8 times, and mix thoroughly. At this time, a white precipitate will appear. Centrifuge at 12000 rpm for 10 minutes.

5) Transfer the supernatant to a new centrifuge tube, add 200 μl endotoxin-free resin solution SW (mix well before use to suspend the resin), place at room temperature for 10 minutes, and invert the centrifuge tube several times during the mixing process.

6) Transfer the obtained solution and resin into the filter column CS, centrifuge at 12000 rpm for 2 min, discard the filter column CS, and collect the solution obtained after centrifugation.

7) Add 700 μl of binding solution PB to the above solution, mix thoroughly, add to the adsorption column CB twice, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid in the collection tube. 8. Add 300 μl binding solution PB, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid in the collection tube.

8) Add 700 μl of rinse solution PW (please check whether absolute ethanol has been added before use), centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid.

9) Repeat the previous step 2 times, and discard the waste liquid.

10) Centrifuge at 12000rpm for 2min to remove the rinse solution as much as possible.

11) Take out the adsorption column CB, put it into a clean centrifuge tube, add 100 μl of elution buffer EB, place at room temperature for 1 min, centrifuge at 12000 rpm for 1 min, add the obtained solution to the centrifugal adsorption column, and centrifuge at 12000 rpm for 1 min.

(12) Enzyme digestion and PCR identification of Anx3 recombinant T vector

Take the bacterial solution as a template for PCR, and the reaction system is 20 μl: 10 μl of 2×Hot-start TaqMasterMix (Tianwei Times Technology Co., Ltd.), 0.5 μl of the upstream and downstream primers of the aforementioned anx3, 8.5 μl of deionized water, and 0.5 μl of the bacterial solution. PCR instrument, denaturation at 95°C for 5 minutes, then cycle, denaturation at 95°C for 1 minute, annealing at 50°C for 1 minute, extension at 72°C for 1.5 minutes, and extension at 72°C for 10 minutes after 30 cycles.

The selected positive clones were identified by restriction endonuclease EcoR I (TaKaRa) after plasmid extraction. The reaction system was 20 μl: EcoR I 1 μl, 10×H Buffer 2 μl, DNA 10 μl, and sterilized water 7 μl. 37°C water bath for 1.5h.

The clones that were positive for enzyme digestion and PCR identification were further subjected to DNA sequencing (Beijing Aoke Biotechnology Co., Ltd.).

(13) Construction of eukaryotic expression vector

Use EcoR I to digest pcDNA3.1/myc-His(-)B plasmid (Invitrogen) (Figure 19), enzyme digestion system 30μl: EcoR I 2μl, 10×H Buffer 3μl, DNA 24μl, sterilized water 1μl, 37℃ water bath After 3.5 hours, the product was recovered with an agarose gel DNA recovery kit (Tianwei Times Technology Co., Ltd.). To prevent self-ligation, dephosphorylate the linearized vector, and the reaction system is as follows: 15 μl linearized vector, 2 μl 10× calf intestinal alkaline phosphatase buffer, 3 μl calf intestinal alkaline phosphatase (Promega), 37 ° C 30min. Afterwards, the dephosphorylated linearized vector was recovered and purified according to DNA Fragment Purification Kit Ver.2.0 (TaKaRa).

Anx3 recombinant T vector was digested with EcoR I, enzyme digestion system 30 μl: EcoR I 2 μl, 10×H Buffer 3 μl, DNA 18 μl, sterilized water 7 μl, 37 ° C water bath for 1.5 h, using agarose gel DNA recovery kit (day for Times Technology Co., Ltd.) to recover the target fragment.

(14) Ligation reaction: Reaction system 10 μl: 10×T ₄ DNA Ligase buffer 1 μl, T4 DNA Ligase (TaKaRa) 1 μl, enzyme-cleaved dephosphorylated pcDNA3.1/myc-His(-)B plasmid 4 μl, target fragment 4 μl, After mixing, place at 16°C overnight.

(15) Transformation of competent cells: add 10 μl of the ligation reaction product to 200 μl of competent cells, the steps are the same as before,

Plate (Amp ⁺ ), pick colonies and add to 5ml LB containing 90μg/ml Amp, 200rpm/min, 37°C overnight. And the experimental steps of enzyme digestion and PCR identification of recombinant plasmids.

(16) Enzyme digestion and PCR identification of Anx3 recombinant eukaryotic expression vector

Take the bacterial liquid as a template for PCR, and add the T7, anx3 downstream primer mixture and T7, anx3 upstream primer mixture (see the primer design section) mixture to identify the forward and reverse connections. The specific steps are the same as above.

After the selected forward and reverse clones were extracted from the plasmid (see above for extracting the plasmid), they were identified by restriction endonuclease EcoRI.

The clones that were positive for enzyme digestion and PCR identification were further subjected to DNA sequencing.

The main process of building pcDB-sense anx3 and pcDB-antisense anx3 is shown in Figure 20.

2. Construct a plasmid (pcDNA3.1-anx3) containing the sense sequence of the annexin A3 (anx 3) target gene

The pUC19-anx3 plasmid (a gift from Professor Céline Raguenes-Nicol, France, I.C.G.M., U332 I.N.S.E.R.M., Paris, France) and the pcDNA3.1(+) plasmid (Invitrogen) (Figure 21) were digested with EcoR I.

20μl system 10×K Buffer 2μl

EcoR I 1μl

                                              

  Deionized water to make up to 7μl

After acting for 3 hours, add 2 μl 10×Loading Buffer (TaKaRa) to terminate the reaction. The product was recovered from the DNA gel, the linear pcDNA3.1(+) plasmid was dephosphorylated, and the solution product was recovered for ligation.

10μl system 10×T ₄ DNA Ligase buffer 1μl

T ₄ DNA Ligase (TaKaRa) 1μl

        Linear pEGFP-N1 plasmid 4 μl

  Target fragment 4μl

After mixing, place at 16°C overnight. The ligation product was transformed, colonies were selected, amplified, identified, and the specific experimental steps were the same as before.

The main process of constructing pcDNA3.1-anx3 is shown in Figure 22.

3. Construction of a plasmid expressing annexin A3 fusion protein (pEGFP-N1-anx3)

The pcDB-sense anx3 and pEGFP-N1 vector plasmids (Clontech, USA) (Figure 23) in the previous step were digested with double restriction enzymes, and they were respectively Xho I and BamH I (TaKaRa).

20μl system 10×K Buffer 2μl

BamH I 1μl

Xho I 1μl

pcDB-sense anx3 16μl

20μl system 10×K Buffer 2μl

BamH I 1μl

Xho I 1μl

                                                     

  Deionized water supplement 20μl

After acting for 3 hours, add 2 μl 10×Loading Buffer to terminate the reaction. DNA gel recovery products were used for ligation.

10μl system 10×T ₄ DNA Ligase buffer 1μl

T ₄ DNA Ligase (TaKaRa) 1μl

Linear pEGFP-N1 plasmid 4 μl

Target fragment 4μl

After mixing, place at 16°C overnight. The ligation product was transformed, colonies were selected, amplified, identified, and the specific experimental steps were the same as before.

The main process of constructing pEGFP-N1-anx3 is shown in Figure 24.

2) Cell transfection

1. Extract plasmid (Qiagen plasmid extraction kit)

(1) Add 5 mL of LB liquid medium containing Apm into two test tubes, respectively insert Escherichia coli containing the plasmids of the sense and antisense genes, and cultivate overnight at 37°C with shaking at 200 rpm/min. Centrifuge 3ml of the overnight cultured bacteria at 4°C, 4000rpm for 10min, and remove the supernatant completely.

(2) Add 300μl Buffer P1 and fully suspend the bacteria with a pipette tip or shaker.

(3) Add 300μl Buffer P2, gently invert up and down 4-6 times to lyse the bacteria, and leave it at room temperature for less than 5 minutes until the solution becomes clear.

(4) Add 300 μl of pre-cooled Buffer P3, immediately invert up and down gently 4-6 times, and ice bath for 5 minutes.

(5) Mix again, centrifuge at 4000rpm for 10min.

(6) Prepare QIAGEN-tip20, add 1ml Buffer QBT to the column, let it flow naturally due to gravity.

(7) Add the supernatant from the fifth step into QIAGEN-tip20 and let it flow naturally.

(8) Add 1ml Buffer QC to QIAGEN-tip20, 4 times in total.

(9) Dilute DNA with 0.8ml Buffer QF. When adding QF, connect the lower port of QIAGEN-tip20 with a clean centrifuge tube and collect the effluent.

(10) Add 0.56ml of isopropanol to the centrifuge tube in the previous step, and immediately centrifuge at 12000 rpm for 30 minutes.

(11) Carefully discard the supernatant, add 1ml of 70% ethanol, centrifuge at 12000rpm for 10 minutes, pour off the ethanol carefully, and dry at room temperature for 5 minutes. Dissolve the pellet with 20 μl TE (pH 8.0).

2. Transfection of Sensitive and Drug-resistant Cells

(1) Digest and subculture the cells in the logarithmic growth phase, inoculate them in a 24-well plate (500 μL) at a density of 0.5×10 ⁶ /ml, culture them in a 37°C, 5% CO ₂ incubator for 24 hours, and then take them out. Confirm that the cell density is 90%-95% confluent.

(2) Add 0.8 μg of plasmid to 50 μL of Opti-MEM ^® I Reduced Serum ((Invitrogen)), and mix gently.

(3) Gently mix Lipofactamine ^TM 2000 ((Invitrogen)) before use, then add Lipofactamine ^TM 2000 2.0 μL to 50 μL Opti-MEM ^® I Reduced Serum, mix well and incubate at room temperature for 5 minutes.

(4) After incubating the liquid in the previous step for 5 minutes, mix it with the diluted plasmid, mix well and incubate at room temperature for 20 minutes.

(5) Cells were replaced with antibiotic-free medium before transfection, then 100 μL of the mixture from the previous step was added and mixed gently. After 4-6h, replace with conventional medium.

(6) Observe the fluorescence intensity and cell transfection at 12, 16, 24, 48, and 72 hours after transfection, take pictures, and calculate the transfection efficiency.

3. Screening of single cell clones stably expressing the target gene

The cell transfection steps were the same as the previous steps 1-5. After 24-48 hours of transfection, the cells were subcultured to a six-well plate at a ratio of 1:10. After 24 hours, G418 was added to a final concentration of 800 μg/ml, and mixed well. G418-containing medium was selected for 3 weeks.

Under the microscope, clones (single, isolated, composed of about 500-1000 cells, uniform in cell shape) were picked by the disk method, inoculated into 24-well plates, and G418 was used to maintain the concentration of 200 μg/ml in the medium.

After the cells grew to the logarithmic growth phase in the 24-well plate, the cells were digested with 0.25% trypsin, seeded into a 6-well plate and continued to be cultured with G418 at a maintenance concentration of 200 μg/ml.

After the cells grew to the logarithmic growth phase in the 6-well plate, the cells were collected and identified by western blot (see the first part of the experiment for specific steps), and the positive clones were picked and frozen. An appropriate amount of cells was retained to continue culture for subsequent experiments.

4. Determination of Drug Resistance of Stable Transfected Cells

After identification by western blot, 2 to 3 cell clones with the highest expression of annexin A3 protein and 2 to 3 cell clones without annexin A3 protein expression were selected respectively, and conventional cell culture was carried out. Cells in the logarithmic growth phase were collected, and IC50 and RI were measured (see the first part of the experiment for specific steps).

(2) Results

1) Construction, screening and identification of positive and antisense plasmids pcDB-sense anx3 and pcDB-antisense anx3

1. Total RNA integrity and purity identification:

The RNA extraction product was electrophoresed on 1% agarose gel, and three RNA bands at 28 s, 18 s, and 5 s were clearly visible under the ultraviolet transilluminator ( FIG. 25 ), which changed from strong to weak, indicating that the RNA was not degraded. The OD value and the highest absorption peak were measured by ultraviolet spectrophotometer. The OD _260nm value was 0.626, the OD _280nm value was 0.337, and the OD _260nm /OD _280nm value was 1.8562, indicating that the RNA was of high purity.

2. Agarose gel electrophoresis results of RT-PCR products: As shown in Figure 26, there is a bright band near the DNA marker 1000bp, which is consistent with the expected target fragment size, which is a 984bp target fragment.

3. Identification of the T vector clone (pGEM-T-anx3): Take the bacterial liquid as a template and perform PCR. It can be seen that there is a bright band near the DNA marker 1000bp in Figure 27, which is consistent with the expected target fragment size. After T vector cloning, positive colonies were selected and digested with the restriction endonuclease EcoRI. The length of the recombinant T vector is about 3kb, and the target fragment of 984bp and the vector fragment of 3kb are produced after enzyme digestion. The electrophoresis results of enzyme digestion identification are shown in Figure 27. After sequencing, the sequence was completely consistent with the known human anx3 sequence in Genbank, and it was confirmed to be a cDNA fragment of anx3 gene.

4. Identification of positive and antisense anx3 eukaryotic expression vectors:

After the pcDB-sense anx3 and pcDB-antisense anx3 recombinant plasmids were transformed into E. coli, positive bacteria were screened, and the bacterial solution was taken as a template for PCR. It can be seen that there is a clear amplification band at 1000 bp of the DNA marker in Figure 29, which is consistent with the expected target fragment size unanimous. Plasmid DNA was extracted from the colonies and identified by EcoRI digestion. The length of the recombinant plasmid containing the insert fragment was about 6.5kb, and the digestion produced a 984bp target fragment and a 5.5kb vector fragment. The digestion results are shown in Figure 29, which is completely consistent with the theoretical calculation. It was further verified by bidirectional sequencing with universal primers, and compared with the cDNA of anx 3 gene in GenBank after sequence splicing, the sequence was 100% consistent (Figure 30). The results showed that the cDNA fragment of anx 3 gene was inserted into the pcDNA3.1/myc-His(-)B vector in forward and reverse directions respectively, and the positive and antisense eukaryotic expression vectors pcDB-sense anx3 and pcDB-antisenseanx3 were successfully constructed. The following is the nucleotide sequence of Annexin A3:

ATGGCATCTATCTGGGTTGGACACCGAGGAACAGTAAGAGATTATCCAGACTTTAG

CCCATCAGTGGATGCTGAAGCTATTCAGAAAGCAATCAGAGGAATTGGAACTGATGAGA

AAATGCTCATCAGCATTCTGACTGAGAGGTCAAATGCACAGCGGCAGCTGATTGTTAAG

GAATATCAAGCAGCATATGGAAAGGAGCTGAAAGATGACTTGAAGGGTGATCCTCTCTGG

CCACTTTGAGCATCTCATGGTGGCCCTAGTGACTCCACCAGCAGTCTTTGATGCAAAGC

AGCTAAAAGAAATCCATGAAGGGCGCGGGAACAAACGAAGATGCCTTGATTGAAATCTTA

ACTACCAGGACAAGCAGGCAAATGAAGGATATCTCTCAAGCCTATTATACAGTATACAA

GAAGAGTCTTGGAGATGACATTAGTTCCGAAACATCTGGTGACTTCCGGAAAGCTCTGT

TGACTTTGGCAGATGGCAGAAGAGATGAAAGTCTGAAAGTGGATGAGCATCTGGCCAAA

CAAGATGCCCAGATTCCTATAAAGCTGGTGAGAACAGATGGGGCACGGATGAAGACAA

ATTCACTGAGATCCTGTGTTTAAGGAGCTTTCCTCAATTAAAACTAACATTTGATGAAT

ACAGAAATATCAGCCAAAAAGGACATTGTGGACAGCATAAAAGGAGAATTATCTGGGCAT

TTTGAAGACTTACTGTTGGCCATAGTTAATTGTGTGAGGAACACGCCGGCCTTTTTAGC

CGAAAGACTGCATCGAGCCTTGAAGGGTATTGGAACTGATGAGTTTTACTCTGAACCGAA

TAATGGTGTCCAGATCAGAAATTGACCTTTTGGACATTCGAACAGAGTTCAAGAAGCAT

TATGGCTATTCCCTATATTCAGCAATTAAATCGGATACTTCTGGAGACTATGAAATCAC

ACTCTTTAAAAATCTGTGGTGGAGATGACTGA

(SEQ ID NO: 1)

Its amino acid sequence is shown in SEQ ID NO: 2

2) Construction, screening and identification of the sense plasmid pcDNA3.1-anx3

The pUC19-anx3 plasmid and the pcDNA3.1(+) plasmid were cut with EcoR I, respectively. A band appeared at 1.3kb after digestion, which was consistent with the expected length of 1339bp. After the fragment was recovered, it was ligated with the linear pcDNA3.1(+) plasmid, and identified by PCR, the band conformed to the target cDNA length (Fig. 31a). Enzyme digestion showed that the length of the recombinant plasmid containing the insert fragment was about 6.7kb, and EcoRI single enzyme digestion produced a 1339bp target fragment and a 5.4kb vector fragment (Figure 31b). It was further verified by bidirectional sequencing with universal primers. After splicing the sequence, it was compared with the cDNA of the anx 3 gene in GenBank, and the sequence was 100% consistent ( FIG. 32 ). The results showed that the cDNA fragment of anx 3 gene was inserted into the pcDNA3.1(+) vector in the forward direction, and the positive-sense eukaryotic expression vector pcDNA3.1-anx3 was successfully constructed.

3) Construction, screening and identification of pEGFP-N1-anx3

Digest pcDB-sense anx3 with Xho I and BamH I, and a bright band appears at 1kb, which is the target fragment of 984bp. After ligation with the linear pEGFP-N1 plasmid, it is about 5.7kb. According to PCR identification, a specific fragment of 984bp can be amplified (Figure 33). Xho I and BamH I double enzyme digestion identification can be seen to produce a 984bp target fragment and a 4.7kb vector fragment (Figure 34). It was further verified by bidirectional sequencing with universal primers, and compared with the cDNA of the anx 3 gene in GenBank after splicing, the sequence was 100% positively consistent (Figure 35). The results showed that the positive-sense eukaryotic expression plasmid pEGFP-N1-anx3 was successfully constructed.

4) Transfection efficiency

In this experiment, various cell lines were transfected by the method of liposome-mediated plasmid DNA, and the fluorescence intensity was observed at 12, 16, 24, 48, and 72 hours after transient transfection, and it was found that the fluorescence intensity was the strongest at the time point of 24 hours. Calculate the transfection efficiency under the microscope, the transfection efficiency of SKOV3 cell line and its drug-resistant cell sub-line is 20%-25%, while the transfection efficiency of A2780 cell line and its drug-resistant cell sub-line is 45%-55% , significantly higher than the SKOV3 group (Figure 36).

5) Screening of positive clones

The sense annexin A3 plasmid was transfected into sensitive cell lines SKOV3 and A2780, and the antisense annexin A3 plasmid was transfected into drug-resistant cell lines SKOV3/CDDP, SKOV3/CBP, A2780/CDDP and A2780/CBP. At the same time, the corresponding empty plasmid was used as a control. G418 screened stable clones, and untreated normal cells were used as controls, and cultured in 800 μg/ml G418 medium for about 8-9 days, the normal cells were all dead, and the pressure was continued to 3w, and a single cell had proliferated to 500 - 1000 cell clones (Figure 37), the positive clones were sequentially selected to 24 and 6-well plates for amplification using the disc method, and the target clones were identified by western blot. As shown in Figure 38, compared with normal cells without any transfection, there was no difference in the expression of annexin A3 in the cells transfected with the empty plasmid. In the cells transfected with sense plasmid, the expression of annexinA3 was significantly up-regulated, and the expression of annexin A3 could not be detected in the cells transfected with antisense plasmid. Among them, the cell clones with the highest and lowest expression levels were selected as the objects of subsequent experiments. Table 10 lists the names of the selected positive cell clones.

6) Determination of drug resistance of cells after stable transfection

After screening and identifying positive clones, drug resistance detection was carried out. The results (Table 11) showed that, compared with transfection of the empty plasmid, the drug resistance index of the sensitive cell line increased by about 2.0 to 3.7 times after transfection with the positive-sense annexin A3 plasmid, especially in the SA4 cell line, and the drug-resistant cell line was significantly higher after transfection. The drug resistance index decreased by about 1.2-2.2 times after the annexinA3 plasmid was used. Among them, the resistance to cisplatin or carboplatin in most of the cells changed significantly after transfection. Although the drug resistance index of SBm to CDDP and ADC6 to CBP only decreased by 1.2-1.4 times, there were statistical differences by t test analysis. It is suggested that the expression of annexin A3 is positively correlated with drug resistance. When annexinA3 is highly expressed, the cell drug resistance increases; when the expression of annexin A3 is inhibited, the cell drug resistance is weakened. form a close relationship.

(3) Conclusion

Using genetic engineering technology, the sense annexin A3 plasmid was transfected into sensitive cell lines SKOV3 and A2780, and the antisense annexin A3 plasmid was transfected into drug-resistant cell lines SKOV3/CDDP, SKOV3/CBP, A2780/CDDP and A2780/CBP. G418 screened stable clones, identified target clones by western blot, and detected drug resistance index after amplification. The results showed that compared with transfection of empty plasmid, the drug resistance index of sensitive cell lines increased by 2 to 4 times after transfection with sense annexin A3 plasmid, and the drug resistance index of drug-resistant cell lines decreased by 2 times after transfection with antisense annexin A3 plasmid about. The results show that annexin A3 is closely related to platinum resistance in epithelial ovarian cancer, and may be involved in the formation of platinum resistance mechanism.

Example 4 Study on the expression of drug resistance-associated protein annexin A3 in epithelial ovarian cancer tissue

(1) Experimental method

1) Ovarian cancer specimen collection: Tumor tissue specimens were collected from ovarian cancer platinum-based chemotherapy-sensitive group and ovarian cancer chemotherapy-resistant group. Strictly standardize the inclusion criteria and match the research samples. From November 2002 to January 2005, 42 specimens were collected from patients with ovarian cancer cytoreductive surgery in the Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Chinese Union Medical University, and confirmed by postoperative pathology to be epithelial ovarian cancer. Inclusion criteria:

1. Primary ovarian epithelial carcinoma diagnosed by pathology.

2. Ovarian cancer chemotherapy-sensitive group: clinical complete remission (CR) was achieved after satisfactory tumor cytoreductive surgery and standard chemotherapy, and the duration was >6 months.

3. Ovarian cancer chemotherapy-resistant group: complete remission (CR) after satisfactory cytoreductive surgery and standard chemotherapy, lasting <6 months; the best tumor response during chemotherapy is PR, SD or PD.

A total of 21 chemotherapy-sensitive tissue samples and 21 drug-resistant tissue samples of ovarian cancer were collected and preserved in paraffin-embedded form, provided by the Pathology Department of Peking Union Medical College Hospital. The clinical data of the corresponding patients are shown in Tables 12 and 13.

The Medical Record Room of Union Medical College Hospital reviewed the medical records of 42 chemotherapy-sensitive and drug-resistant patients, and found out all the intraoperative specimen slices according to the pathology number. Tumor parenchyma-dominated sections were used to search for corresponding cancer tissue wax blocks for immunohistochemistry.

2) Immunohistochemical staining

1. The paraffin-embedded tumor tissue was serially sectioned with a thickness of about 4um. After cutting out the paraffin wax slices, soak them in 30% alcohol and 50°C water for a while to fully expand them. Transfer to a glass slide and bake at 75°C for 45 minutes.

2. Sequential xylene deparaffinization (15min×3). Soak in serial concentrations of ethanol (100%, 95%, 80%) sequentially for a while, rinse with distilled water twice, and soak in PBS for 15-30 min.

3. Repair the tissue antigens by various methods in advance, and choose the antigen restoration method with the best effect. After testing, AnnexinA3 was repaired by microwave.

4.3% hydrogen peroxide blocked for 10 minutes.

5. Add the primary antibody, put the slice into a wet box, incubate at room temperature for 75min, wash with PBS for 5min×3 times.

6. Add the secondary antibody to the automatic immunohistochemical staining instrument, put the slices into a wet box, incubate at room temperature for 45 minutes, and wash with PBS for 5 minutes×3 times.

7.DAB color development for 1-5min, controlled by light microscope. PBS, rinse with distilled water.

8. Hematoxylin restaining: Soak in hematoxylin for 1 min, rinse with distilled water, digest with hydrochloric acid alcohol for a while, soak in dilute ammonia for 1 min, rinse with distilled water.

9. Soak in serial concentration ethanol (80%, 95%, 100%) for a while, and xylene for a while (×2).

10. Neutral resin sealant.

3) Image reading, judging criteria and positive and negative control

All slides were read by pathologists without knowing any clinical data.

Criterion for judging: the staining is positive when the cytoplasm is brown. According to the percentage of colored tumor cells in all tumor cells, <5% is (-), 5-25% is (+), 25-50% is (++), 50%-75% is (+++), >75% is (++++), randomly select 10 visual fields, and the average value is taken as the final result.

Positive control: annexinA3 uses liver cells as a positive control, and the positive part is the cytoplasm.

Negative control: replace primary antibody with rabbit serum before immunization.

4) Statistical analysis

Use SPSS11.5 software to carry out the following statistical analysis:

The rank sum test of rank data was used to evaluate whether there was a significant difference in the expression of annexin A3 between chemotherapy-sensitive and drug-resistant patients.

(2) Results

The average age of ovarian cancer chemotherapy-sensitive group was 50.29 years old, and the average age of chemotherapy-resistant group was 55.29 years old. There was no significant difference in age between the two groups (P=0.265). There was no significant difference in ovarian cancer stage between the two groups (P=0.141).

Annexin A3 is mainly expressed in the cytoplasm, and partially expressed in the nucleus, as shown in Figure 39. The rank sum test of graded data was used to compare the statistical results of patients in the chemotherapy-sensitive group and the drug-resistant group, showing that U value = 139, P value = 0.035 < 0.05, indicating that there is a significant difference between the expression levels of annexinA3 in chemotherapy-sensitive patients and drug-resistant patients The expression level of annexin A3 in chemotherapy-resistant patients was higher than that in sensitive patients, see Tables 14 and 15. The clinical results show that annexin A3 is closely related to the formation of platinum resistance in epithelial ovarian cancer.

(3) Conclusion

According to the immunohistochemical staining study on the ovarian cancer tissues of 21 chemotherapy-sensitive patients and 21 drug-resistant patients, the results showed that there was a significant difference in the expression of annexin A3 between the two groups (P=0.035), suggesting that annexin A3 is a platinum Drug resistance-related proteins are expected to become markers for predicting platinum resistance and prognosis of ovarian cancer, and may become new targets for reversing clinical drug resistance.

Table 1. RT-PCR primers, product length, annealing temperature name sequence product (bp) Annealing temperature (℃) β-actin ACACTGTGCCCATCTACGAGGAGGGGCCGGACTCGTCATACT 621 58 MDR-1 CCCATCATTGCAATAGCAGGGTTCAAACTTCTGCTCCTGA 157 58 MRP-1 ACCAAGACGTATCAGGTGGCCTGTCAGGTTCCAGCTCCTC 428 58 LRP-1 ACAACTACTGCGTGATTCTCGGGTCTTGACATCCTGCACATA 350 58 GST-pi GGTGGTGACCGTGGAGACTCATGGATCAGCAGCAAG 397 58

Table 2. Cell cycle distribution of SKOV3 and drug-resistant cells

^* P<0.05, ^** P<0.01, compared with SKOV3 cell cycle SKOV3 SKOV3/CDDP-P SKOV3/CDDP-80 G ₀ /G ₁ (%) 46.15±3.26 60.74±2.36 ^** 65.41±3.88 ^** S(%) 40.30±2.68 27.76±1.69 ^* 26.74±1.93 ^** G ₂ /M(%) 13.55±0.59 11.50±0.70 ^* 7.85±1.57 ^*

Table 3. Primer sequences, fragment lengths and annealing temperatures for real-time quantitative PCR name sequence product (bp) Annealing temperature (℃) SEQ ID NO: annexin A3 TGAAGGGTATTGGAACTGATGTGAGAAGAAGTAAGGTGGAGC 254 52 34 destrin GTAAATGCTCCACACCAGAAGGCATCATACAAAGCATAGCGA 199 52 56 cofilin 1 TATGAGACCAAGGAGAGCAAGCTTGACCTCCTCGTAGCAGTT 148 52 78 GSTO1-1 AAGCATACCCAGGGAAGAAGCTGCCATCCACAGTTTCAGTTT 334 52 910 ikB AGAGCAAAGCTTGATAACAATGAAAAATGTAAACCTGTAGAC 284 52 1112 beta-actin AAACTACCTTCAACTCCATCAAACTAAGTCATAGTCCGCCTA 319 52 1314

Table 4. IC50 and RI values of six cell lines, "-": IC50 value not determined cell line IC50(μmol/L) RI CDDP CBP SKOV3 6.67±2.58 80.79±10.82 1.00 SKOV3/CDDP 27.24±8.60 - 4.12 SKOV3/CBP - 242.40±20.36 3.00 A2780 1.95±0.69 50.26±5.38 1.00 A2780/CDDP 6.83±1.41 - 3.50 A2780/CBP - 175.90±15.27 3.50

Table 5. Differentially expressed proteins between SKOV3/CDDP and SKOV3 cells serial number protein name protein sequence number Isoelectric point Protein Molecular Weight (Da) Peptide Coverage (%) protein score Express theoretical value observed value theoretical value observed value match total 01 Villin 2 gi|46249758 5.94 6.48 69199 75258 47 69 60 328 ↓ 04 Stomatin (EPB72)-like 2 gi|7305503 6.88 5.50 38510 43203 19 26 53 177 ↓ 07 NADP-dependent isocitrate Dehydrogenase (IDHc) gi|3641398 6.34 6.89 46659 44484 25 41 63 321 ↓ 12 Annexin A4 gi|1703319 5.84 5.94 35860 33421 twenty one 38 58 145 ↑ 16 ATP synthase, H+transporting, mitochondrial F0 complex, subunit d isoform a gi|5453559 5.21 5.19 18480 24953 10 17 72 123 ↑ 17 Activated RNA polymerase IItranscription cofactor 4 gi|19923784 9.6 5.21 14356 18564 10 twenty four 55 137 ↑ twenty one Heat shock 70kDa protein 1A gi|5123454 5.48 5.56 69995 66358 32 56 60 251 ↑ 25 Annexin A3 gi|47115233 5.76 5.76 36452 35643 19 36 55 238 ↑ 26 Proteasome (prosome, macropain) subunit, alpha type, 1 gi|30582133 6.15 6.65 29579 31958 16 55 51 101 ↓ 27 Prohibitin gi|46360168 5.57 5.62 29802 29657 14 46 67 203 ↑ 28 Heat shock protein 27 gi|662841 7.83 5.71 22313 26385 16 57 73 221 ↑ 34 Cofilin 1 (non-muscle) gi|5031635 8.22 6.57 18491 18698 7 20 46 113 ↓ 54 Glutathione-S-transferase omega 1 gi|4758484 6.23 6.51 27548 33568 11 35 39 84 ↑ 67 Destrin gi|5802966 8.84 6.90 18493 19201 4 6 47 65 ↑

Table 6. Differentially expressed proteins between SKOV3/CBP and SKOV3 cells serial number protein name protein sequence number Isoelectric point Protein Molecular Weight (Da) Peptide Coverage (%) protein score Express theoretical value observed value theoretical value observed value match total 04 Stomatin (EPB72)-like 2 gi|7305503 6.88 5.50 38510 43203 19 26 53 177 ↑ 07 NADP-dependent isocitrated hydrogenase (IDHc) gi|3641398 6.34 6.89 46659 44484 25 41 63 321 ↓ 09 Annexin Al gi|54696696 6.57 6.68 38690 39621 twenty two 34 63 228 ↓ 11 Unnamed protein product gi|7022776 8.99 5.50 21244 33852 15 20 35 95 ↓ 13 Lactamase, beta 2 gi|7705793 6.32 6.83 32785 32901 8 33 34 68 ↓ 14 Phosphoglycerate mutase 1 gi|38566176 6.67 6.69 28802 29587 13 30 48 252 ↑ 15 Prosome beta-subunit; HSBpros26 gi|551547 5.70 5.74 25893 28356 11 17 48 108 ↑ 18 Stathmin 1 gi|15680064 5.76 5.49 17326 19068 9 14 51 114 ↑ 19 Fatty acid binding protein5 gi|30583737 6.60 6.47 15155 16234 15 30 68 126 ↓ twenty one Heat shock 70kDa protein 1A gi|5123454 5.48 5.56 69995 66358 32 56 60 251 ↓ 25 Annexin A3 gi|47115233 5.76 5.76 36452 35643 19 36 55 238 ↑ 28 Heat shock protein 27 gi|662841 7.83 5.72 22313 28985 16 57 73 221 ↓ 34 Cofilin 1 (non-muscle) gi|5031635 8.22 6.57 18491 18698 7 20 46 113 ↓ 40 Peroxiredoxin 1 gi|55959887 8.27 8.06 18964 25487 twenty four 57 87 262 ↑ 47 ECH1 protein gi|16924265 8.47 6.49 35735 33069 5 7 twenty four 69 ↑ 67 Destrin gi|5802966 8.84 6.90 18493 19201 4 6 47 65 ↑

Table 7. Differentially expressed proteins between A2780/CDDP and A2780 cells serial number protein name protein sequence number Isoelectric point Protein Molecular Weight (Da) Peptide Coverage (%) protein score Express theoretical value observed value theoretical value observed value match total 07 NADP-dependent isocitrated hydrogenase (IDHc) gi|3641398 6.34 6.89 46659 44484 25 41 63 321 ↓ 08 Aldehyde Reductase gi|1633300 6.34 6.78 36419 40188 18 29 53 188 ↑ 10 Aldo-keto reductase family 1, member B1 gi|13529257 6.6 6.90 35801 39018 11 twenty three 25 158 ↑ 25 Annexin A3 gi|47115233 5.76 5.76 36452 35643 19 36 55 238 ↑ 27 Prohibitin gi|46360168 5.57 5.62 29802 29657 14 46 67 203 ↓ 33 Splicing factor, arginine/serine-rich 3 gi|56417681 10.12 6.19 14194 24015 8 26 54 124 ↑ 34 Cofilin 1 (non-muscle) gi|5031635 8.22 6.57 18491 18698 7 20 46 113 ↑ 51 Lactate dehydrogenase B gi|4557032 5.71 5.92 36615 39120 12 14 twenty four 99 ↑ 54 Glutathione-S-transferase omega 1 gi|4758484 6.23 6.51 27548 33568 11 35 39 84 ↑ 55 Peroxiredoxin 6 gi|4758638 6.00 6.17 25019 27933 10 14 47 103 ↑ 56 dUTP pyrophosphate gi|4503423 6.15 5.75 17737 21532 5 7 48 81 ↓ 63 Purine nucleotide phosphorylase gi|4557801 6.45 6.70 32127 32085 twenty one 31 69 199 ↑ 67 Destrin gi|5802966 8.84 6.90 18493 19201 4 6 47 65 ↑ 68 Triosephosphate isomerase 1 gi|17389815 6.45 6.48 26625 28954 9 11 47 138 ↓ 81 T-complex protein 1, gamma subunit (TCP-1-gamma) (CCT-gamma) gi|20455521 6.10 6.47 60364 65539 31 43 47 242 ↑

Table 8. Differentially expressed proteins between A2780/CBP and A2780 cells serial number protein name protein sequence number Isoelectric point Protein Molecular Weight (Da) Peptide Coverage (%) protein score Express theoretical value observed value theoretical value observed value match total 07 NADP-dependent isocitrated hydrogenase (IDHc) gi|3641398 6.34 6.89 46659 44484 25 41 63 321 ↓ 18 Stathmin 1 gi|15680064 5.76 5.49 17326 19068 9 14 51 114 ↓ 25 Annexin A3 gi|47115233 5.76 5.76 36452 35643 19 36 55 238 ↑ 31 UMP-CMPK gi|33150592 5.44 5.64 22208 26694 11 29 55 147 ↑ 32 Adeninephosphoribosyltransferase gi|31415697 5.78 5.56 19595 24363 10 26 72 151 ↑ 34 Cofilin 1 (non-muscle) gi|5031635 8.22 6.57 18491 18698 7 20 46 113 ↑ 47 ECH protein 1 gi|16924265 8.47 6.49 35735 33069 5 7 twenty four 69 ↓ 54 Glutathione-S-transferase omega 1 gi|4758484 6.23 6.51 27548 33568 11 35 39 84 ↑ 56 dUTP pyrophosphatase gi|4503423 6.15 5.75 17737 21532 5 7 48 81 ↓ 67 Destrin gi|5802966 8.84 6.90 18493 19201 4 6 47 65 ↑ 82 J-type co-chaperone HSC20 gi|41388876 7.59 6.26 27405 30583 7 14 33 71 ↓ 83 KIAA0158 gi|40788885 6.05 6.57 42120 43307 15 36 46 110 ↑

Table 9. Differentially expressed proteins shared by more than three drug-resistant cells identified by MALDI-TOF-MS protein name protein sequence number Isoelectric point Protein Molecular Weight (Da) Peptide Coverage (%) protein score Differential expression bits theoretical value observed value theoretical value observed value match total ikB gi|3641398 6.34 6.89 46659 44484 25 41 63 321 2～9 Annexin A3 gi|47115233 5.76 5.76 36452 35643 19 36 55 238 3～20 Cofilin 1 gi|5031635 8.22 6.57 18491 18698 7 20 46 113 2～10 GSTO1-1 gi|4758484 6.23 6.51 27548 33568 11 35 39 84 2～5 Destrin gi|5802966 8.84 6.90 18493 19201 4 6 47 65 2～3

Table 10. Nomenclature of target clones selected after western blot identification cell line transfection plasmid name cell line transfection plasmid name SKOV3 pcDNA3.1(+) SM A2780 pcDNA3.1(+) Am SKOV3 pcDNA3.1-anx3 SA4 A2780 pcDNA3.1-anx3 AA4 SKOV3/CDDP pcDNA3.1(+) SDm A2780/CDDP pcDNA3.1(+) ADm SKOV3/CDDP pcDB-antisenseanx3 SDR6 A2780/CDDP pcDB-antisenseanx3 ADR6 SKOV3/CBP pcDNA3.1(+) SB A2780/CBP pcDNA3.1(+) AB SKOV3/CBP pcDB-antisenseanx3 SBR6 A2780/CBP pcDB-antisenseanx3 ABR6

Table 11. Detection of drug resistance of target clones after stable transfection cell line IC50(cisplatin) IC50 (carboplatin) IC50(μM) RI IC50(μM) RI SM 6.12±1.67 1.00 87.73±20.89 1.00 SA4 22.83±3.74** 3.73 213.16±28.4** 2.43 SDm 16.56±3.12 1.00 242.73±19.11 1.00 SDR6 7.38±2.23** 0.45 122.59±15.71** 0.51 SB 10.98±2.44 1.00 169.66±16.93 1.00 SBR6 9.13±1.83* 0.83 102.28±13.17** 0.60 Am 1.81±0.45 1.00 60.42±11.73 1.00 AA4 3.69±1.07* 2.04 107.79±13.57** 1.78 ADm 4.45±1.15 1.00 125.28±19.18 1.00 ADR6 2.34±0.62* 0.53 91.81±13.93* 0.73 AB 9.79±2.01 1.00 135.7±15.37 1.00 ABR6 6.02±2.18* 0.61 62.13±10.54** 0.46

Table 12. Clinical data of 21 patients with ovarian cancer chemotherapy sensitive serial number age organization type installments S1 57 clear cell carcinoma IIIc S2 63 clear cell carcinoma IIIc S3 56 Serum breast cancer IIIc S4 50 Serum breast cancer IIc S5 40 Serum breast cancer IIIc S6 49 Serum breast cancer IIIc S7 42 Serum breast cancer IIIc S8 46 Serum breast cancer IIIc S9 53 Serum breast cancer IIIc S10 67 Serum breast cancer IIIb S11 57 Serum breast cancer IIIc S12 49 Serum breast cancer IV S13 48 Serum breast cancer IIa S14 47 milk low IIIc S15 62 endometrioid carcinoma IIIc S16 39 endometrioid carcinoma IIc S17 44 endometrioid carcinoma IIIc S18 49 endometrioid carcinoma IIc S19 58 endometrioid carcinoma IIIc S20 42 endometrioid carcinoma IIIc S21 38 mucinous carcinoma IIIc

Table 13. Clinical data of 21 patients with ovarian cancer chemotherapy resistance serial number age organization type installments R1 64 clear cell carcinoma IIIc R2 61 clear cell carcinoma IIIc R3 51 clear cell carcinoma IV R4 63 Serum breast cancer IIIc R5 48 Serum breast cancer IIIc R6 52 Serum breast cancer IIIc R7 52 Serum breast cancer IIc R8 52 Serum breast cancer IIIc R9 61 Serum breast cancer IIIc R10 54 Serum breast cancer IIIc R11 65 Serum breast cancer IIIc R12 75 Serum breast cancer IIIc R13 51 Serum breast cancer IIIc R14 42 Serum breast cancer IIIb R15 51 Serum breast cancer IIIc R16 59 transitional cell carcinoma IIIc R17 52 transitional cell carcinoma IIIc R18 55 transitional cell carcinoma IIIc R19 54 endometrioid carcinoma IIIc R20 53 endometrioid carcinoma IIIc R21 52 mucinous carcinoma IIIc

Table 14. Staining results of chemotherapy-sensitive and drug-resistant tissue samples of 42 cases of ovarian cancer serial number Annexin A3 serial number Annexin A3 S1 4+ R1 4+ S2 3+ R2 4+ S3 3+ R3 3+ S4 2+ R4 3+ S5 2+ R5 3+ S6 2+ R6 3+ S7 2+ R7 3+ S8 1+ R8 2+ S9 1+ R9 2+ S10 1+ R10 2+ S11 1+ R11 2+ S12 1+ R12 2+ S13 1+ R13 2+ S14 1+ R14 1+ S15 - R15 1+ S16 - R16 1+ S17 - R17 1+ S18 - R18 1+ S19 - R19 1+ S20 - R20 - S21 - R21 -

Table 15. Comparison of the expression results of Annexin A3 in ovarian cancer chemotherapy-sensitive and drug-resistant tissues Annexin A3 expression level sensitive (example) drug resistance (example) - 7 2 1+ 7 6 2+ 4 6 3+ 2 5 4+ 1 2 total twenty one twenty one

Sequence Listing

<110> Peking Union Medical College Hospital, Chinese Academy of Medical Sciences

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Claims (26)

1. A method for regulating the drug resistance of platinum-based chemotherapy drugs in cancer, comprising changing the expression of annexin A3 in cancer cells, wherein the cancer is preferably ovarian cancer, more preferably epithelioid ovarian cancer, and the platinum-based chemotherapy drugs are preferably cis platinum or carboplatin. 2. The method of claim 1 for modulating drug resistance in cancer cells in vitro or for modulating drug resistance in cancer patients in vivo. 3. The method of claim 1, by increasing the expression of annexin A3 in cancer cells and improving the tolerance of cancer cells to platinum chemotherapy drugs, preferably, by expressing annexin A3 genes in cancer cells and increasing annexin A3 in cells Expressing, again preferably wherein the content of annexin A3 in said cells is increased by using a plasmid expressing a positive-sense annexin A3 gene. 4. The use of a medicament capable of increasing the expression of annexin A3 in cancer cells in the preparation of a drug that improves the resistance of cancer to platinum-based chemotherapy drugs, wherein preferably the medicament is selected from: a polynucleotide encoding annexin A3 protein, comprising A DNA construct or an expression vector of a polynucleotide encoding annexin A3 protein, wherein the cancer is preferably ovarian cancer, more preferably epithelioid ovarian cancer, and the platinum chemotherapy drug is preferably cisplatin or carboplatin. . 5. The method of claim 1, by reducing and/or inhibiting the expression of annexin A3 in cancer cells and reducing the tolerance of cancer to platinum chemotherapy drugs, preferably, by expressing the antisense nucleic acid of annexin A3 in cancer cells and /or specific to the ribozyme of annexin A3 to reduce and/or inhibit the expression of annexin A3 in the cell, preferably wherein the content of annexin A3 in the cell is reduced by using a DNA construct or an expression vector expressing antisense annexin A3. 6. An antisense nucleotide that inhibits the expression of annexin A3. 7. the antisense nucleotide of claim 6, it comprises the nucleotide sequence of all or part of any sequence that can specifically bind to annexin A3 polynucleotide sequence or its complementary sequence, preferably said antisense nucleotide contains A sequence region that is complementary, more preferably substantially complementary, even more preferably fully complementary to one or more portions of the annexin A3 polynucleotide, more preferably, the antisense nucleotide comprises a sequence region that is compatible with the full-length annexin A3 polynucleotide A fully complementary DNA sequence, preferably, the antisense nucleotide is DNA or RNA or derivatives thereof, most preferably, the antisense nucleotide is contained in an expression plasmid capable of expressing it, preferably eukaryotic expression in the plasmid. 8. A ribozyme that specifically cuts annexin A3 mRNA. 9. The ribozyme of claim 8, which has a specific substrate binding site complementary to one or more annexin A3 RNA regions, and it has a core that imparts the molecular RNA cleavage activity in or around the substrate binding site Nucleotide sequence, preferably said ribozyme is selected from: hammerhead type ribozyme, hairpin type ribozyme, hepatitis D virus type ribozyme, I type intron type ribozyme or RNasePRNA type ribozyme or Neurospora It belongs to VS RNA type ribozyme. 10. The application of the medicament capable of reducing and/or inhibiting the expression of annexin A3 in cancer cells in the preparation of a drug resistant to platinum-based chemotherapeutic drugs for cancer, wherein the medicament is preferably selected from: the reverse of claim 6 or 7 Sense nucleotides and the ribozyme according to claim 8 or 9, wherein the cancer is preferably ovarian cancer, more preferably epithelioid ovarian cancer, and the platinum chemotherapeutic drug is preferably cisplatin or carboplatin. 11. The cancer cell that has changed the tolerance to platinum-based chemotherapeutics by the method of claim 1, preferably, said cell comprises the expression vector expressing sense annexin A3 or antisense annexinA3, preferably, said vector is a plasmid , wherein the cancer is preferably ovarian cancer, more preferably epithelioid ovarian cancer, and the platinum chemotherapy drug is preferably cisplatin or carboplatin. 12. A method of screening an agent capable of changing the drug resistance of cancer to a platinum-based chemotherapeutic drug, wherein the cancer is preferably ovarian cancer, more preferably epithelioid ovarian cancer, and the platinum-based chemotherapeutic drug is preferably cisplatin or carbane Platinum, the method includes the steps of: providing a candidate agent; contacting the candidate agent with cancer cells; detecting the expression of annexin A3 in the cancer cells; comparing the detected expression amount with a predetermined threshold. 13. A medicament obtained by the screening method of claim 12. 14. A method for detecting the drug resistance of cancer cells to platinum chemotherapy drugs, wherein the cancer is preferably ovarian cancer, more preferably epithelioid ovarian cancer, and the platinum chemotherapy drugs are preferably cisplatin or carboplatin, so The method includes detecting the expression level of annexin A3 in cancer cells, and comparing the obtained expression level with a predetermined threshold, thereby indicating the resistance of ovarian cancer cells to platinum chemotherapy drugs. 15. A method for predicting or prognosing the therapeutic effect of platinum-based chemotherapy drugs in cancer patients, wherein the cancer is preferably ovarian cancer, more preferably epithelioid ovarian cancer, and the platinum-based chemotherapy drugs are preferably cisplatin or carboplatin , the method comprises detecting the expression amount of annexin A3 in the tumor tissue of a cancer patient, and comparing the obtained amount with a predetermined threshold, thereby predicting or prognosing the therapeutic effect of administering a platinum chemotherapy drug to the patient, that is, Predicting resistance to platinum-based chemotherapy agents in patients. 16. The method of claim 12, 14 or 15, wherein the step for detecting the expression level of annexin A3 is implemented by detecting the mRNA level or protein level of annexin A3; wherein, preferably, the detection at the protein level is carried out by antibodies or Oligonucleotide aptamers are used, preferably using antibodies; and detection at the mRNA level is preferably achieved by Northern blotting or PCR. 17. An antibody specifically binding to annexin A3 or an antigen-binding fragment thereof, wherein the antibody may be a monoclonal antibody or a polyclonal antibody, preferably a neutralizing antibody. 18. An oligonucleotide aptamer specifically binding to annexin A3 protein, preferably the oligonucleotide aptamer is DNA, preferably, the oligonucleotide aptamer is connected or labeled with other functional groups Or molecules such as thiols, amino groups, radioactive isotopes, fluorescein, biotin, or enzymes, etc. 19. The use of a medicament capable of detecting the expression of annexin A3 in the preparation of a detection or diagnostic kit for detecting resistance to platinum-based chemotherapy drugs in cancer cells or diagnosing drug resistance to platinum-based chemotherapy drugs in cancer patients, wherein the cancer It is preferably ovarian cancer, more preferably epithelioid ovarian cancer, and the platinum-based chemotherapeutic drug is preferably cisplatin or carboplatin, wherein preferably, the agent is selected from: the antibody or its fragment according to claim 17, the oligonucleotide according to claim 18 Nucleotide aptamers, nucleic acid probes and primers specific to the annexin A3 gene, or combinations thereof, more preferably, the agent is an antibody to the annexin A3 protein, preferably a monoclonal antibody. 20. A method of treating a cancer patient, comprising the steps of: a) reducing the patient's tolerance to a platinum-based chemotherapeutic drug by the method of the present invention for altering the resistance of a cancer to a platinum-based chemotherapeutic drug; and b) administering platinum to said patient Chemotherapy drugs, optionally, before step (a), detect the resistance of cancer patients to platinum-based chemotherapy drugs to determine drug-resistant patients, wherein the cancer is preferably ovarian cancer, more preferably epithelioid ovarian cancer, the The platinum chemotherapy drug is preferably cisplatin or carboplatin. 21. A method for treating a cancer patient, said method comprising: determining the patient's platinum-based chemotherapeutic drug resistance according to the present invention, and adjusting the patient's platinum-based chemotherapeutic drug regimen, wherein the cancer is preferably ovarian cancer, more preferably For epithelioid ovarian cancer, the platinum chemotherapy drug is preferably cisplatin or carboplatin. 22. A composition comprising the medicament of claim 4, 10, 13 or 19, preferably, the composition is used for diagnosing and/or changing the resistance of a cancer to a platinum-based chemotherapeutic drug, wherein the cancer is preferably is ovarian cancer, more preferably epithelioid ovarian cancer, and the platinum chemotherapy drug is preferably cisplatin or carboplatin. 23. The composition of claim 22, wherein the medicament is preferably selected from the group consisting of: the antisense nucleotide of claim 6 or 7, the ribozyme of claim 8 or 9, the antibody of claim 17, the oligonucleotide of claim 18 One or any combination of nucleotide aptamers. 24. A method for establishing a platinum-based chemotherapeutic drug-resistant cancer cell line, comprising: inducing cancer cells with a high-dose platinum-based chemotherapeutic drug shock. 25. The aforementioned invention, wherein the cancer is a cancer that can be treated with platinum-based chemotherapy drugs, such as ovarian cancer, breast cancer, lung cancer, testicular tumor, head and neck cancer, osteosarcoma, melanoma, esophageal cancer, etc., preferably ovarian cancer Cancer, more preferably human serous ovarian cancer and epithelioid ovarian cancer, including endometrioid carcinoma, seromammary carcinoma, adenocarcinoma, transitional cell carcinoma, endometrial carcinoma, clear cell carcinoma. 26. The aforementioned invention, wherein the platinum-based chemotherapeutic drug is cisplatin, carboplatin, oxalplatin, nedaplatin, roplatin, etc., preferably cisplatin and carboplatin.

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