CN101768214B - Human tumor marker-Tim17 polypeptide and application thereof - Google Patents

Abstract

The invention belongs to the fields of biotechnology and medicine, and relates to a new human tumor marker-Tim17 polypeptide, a polynucleotide sequence encoding Tim17 and a method for producing the protein through recombinant technology. The present invention also relates to the application of Tim17 protein and its coding sequence, and the pharmaceutical composition containing Tim17 protein antagonist. Experiments have shown that the Tim17 polypeptide, its coding sequence, and the expression product of the gene are almost undetectable in normal cells and tissues, but are highly expressed in tumor cells and tissues, and the expression level is comparable to that of tumors. Grade, degree of differentiation and prognosis were significantly positively correlated. The results showed that Tim17 was a specific tumor marker. It can be used as a marker for the prevention and early detection of breast cancer, lung cancer, liver cancer, colon cancer, bladder cancer, prostate cancer or lymphoma or a target for tumor treatment.

Description

A human tumor marker-Tim17 polypeptide and its application

technical field

The invention belongs to the fields of biotechnology and medicine. Specifically, it relates to a new human tumor marker-Tim17 polypeptide (translocase of inner mitochondrial membrane 17), a polynucleotide sequence encoding Tim17 and a method for producing the protein through recombinant technology. The present invention also relates to the use of Tim17 protein and its coding sequence, such as for tumor prevention, early detection, prognosis judgment, and pharmaceutical composition containing Tim17 protein antagonist (such as antibody and antisense nucleic acid, RNAi, etc.).

Background technique

Biomarkers refer to physical, chemical, or biological indicators that can be objectively measured, indicating normal physiological or pathological processes, or the body's response to drugs. One of the most critical issues in tumor research is to find markers for early detection, diagnostic typing, treatment monitoring and prognosis, and as drug targets. During the transformation of cells from a normal state to a tumor, a series of changes occur in the intracellular protein expression profile. Tumor markers are a class of substances synthesized and released by tumor cells or released by the host in response to tumors during the occurrence and development of tumors. They have certain specificity and sensitivity, and can be used as monitoring indicators in diagnosis and treatment.

At present, tumor markers that have been used clinically are still quite lacking. The review standards for markers vary slightly from country to country. In terms of the application of tumor markers in my country, we mainly refer to the standards of Western developed countries such as the United States. Taking the United States as an example, every year ASCO (American Society of Clinical Oncology) will make guiding recommendations on biomarkers used in clinical oncology. For example, markers that can be used for breast cancer in 2007 include ER/PR, HER2, uPA/PAI1, and multi-parameter gene expression analysis methods (Oncotype DM, MammaPrint, etc.), and provide detailed recommended conditions and scope. The rest such as Ki67, Cyclin D, Cyclin E, p27, p21, proteome analysis, etc. are not recommended due to insufficient evidence (see ASCO web page). It can be seen that while acknowledging the role of these markers in improving patient diagnosis and treatment, the current morbidity and mortality of cancer patients are still on the rise. This phenomenon requires the discovery and clinical application of more and better tumor markers.

Contents of the invention

The object of the present invention is to provide a novel human tumor marker Tim17 polypeptide SEQ ID NO: 1, nucleic acid (RNA) SEQ ID NO: 5, and fragments, analogs, and derivatives thereof.

Another object of the present invention is to provide coding above-mentioned RNA, the deoxynucleic acid sequence (DNA) SEQ ID NO of polypeptide: 6.

Another object of the present invention is to provide the preparation method of the above-mentioned RNA and polypeptide, as well as the use of the RNA, polypeptide and its coding sequence.

Said RNA can be produced by means of PCR. Specifically, the primer pair in SEQ ID NO: 3 can be used to obtain the RNA product whose sequence is SEQ ID NO: 5 by polymerase chain reaction. The polypeptide can be produced by chemical synthesis. Both the RNA and the peptide can help detect the presence of tumors.

The present invention discovers a new gene that can be used as a tumor marker after extensive and in-depth research. The expression products of this gene, including RNA SEQ ID NO: 4 and protein SEQ ID NO: 2, are almost undetectable in normal cells and tissues, but highly expressed in tumor cells and tissues. And the expression level is significantly positively correlated with tumor grade, degree of differentiation, and patient prognosis. The results showed that Tim17 is a specific tumor marker. The present invention has been accomplished on this basis.

Specifically, the present invention obtains a series of differentially expressed proteins by systematically comparing breast cancer cell lines with different malignant degrees from the same patient. Timm17A is one of the novel tumor-specific expressed proteins. One of its polypeptides, SEQ ID NO: 1, was specifically detected by stable isotope-labeled mass spectrometry, and its expression level in tumors was 4.8 times higher than that in normal cells. By quantitative RT-PCR method, the results showed that the RNA expression of Timm17A in the tumor was also significantly higher than that of the normal control. At the same time, through antibody verification, it was found that the protein of Tim17 (including A and B subtypes, and the antibody did not distinguish between these two subtypes) was also on the rise in tumors (Fig. 1A, B). Furthermore, by immunohistochemical method, the present invention tested the tissues of 43 normal and breast cancer patients (Fig. 1C, Table 1). The expression of Tim17 is shown in breast diseases (Hyperplasia, breast hyperplasia); the expression peaks in early breast cancer patients (DCIS, ductal tumor in situ). The above experimental evidence indicates that Tim17 has a potential role as a marker for early detection of breast cancer.

In order to verify the above experimental results, the present invention has carried out the work of database data mining. In the Oncomine ( www.oncomine.org ) database, RNA microarray chip data of a large number of tumor cases are stored. The present invention searches Timm17A gene and arranges data, wherein, the comparison result of breast cancer and normal tissue shows that the expression level of Timm17A in breast cancer is significantly higher than that of normal control (Figure 2); In the expression correlation analysis of , a significant positive correlation was shown (Fig. 3). More meaningfully, in the correlation analysis between the patient's prognosis and the expression of Timm17A, a significant positive correlation was also shown (Fig. 4); at the same time, another subtype of Timm17, the Timm17B gene, also showed the above results. The above results indicate that both subtypes of Tim17 can be potential markers for tumor discovery (early detection), diagnosis (grading), and prognosis.

In order to observe whether the high tumor expression of Tim17 is only limited to breast cancer, the present invention also performed similar data mining analysis on other tumors in the Oncomine database. The results showed that in lung cancer, liver cancer, intestinal cancer, bladder cancer, prostate cancer, and lymphoma, there is a phenomenon of high expression in this kind of tumor (Fig. 5). The analysis results confirmed that Tim17 is a broad-spectrum tumor marker.

According to the overexpression of Tim17 in the tumor, the present invention checks whether inhibiting its expression can inhibit the growth of the tumor: the shRNA targeting Tim17 is designed and synthesized, and it is cloned into a lenti-viral vector capable of inducible expression . The results showed that inhibiting the expression of Tim17 could significantly inhibit tumor growth. The results further confirm that Tim17 can be used as a target for tumor therapy.

Description of drawings

Figure 1 Tim17 expression in breast cancer cell lines and tissues,

Among them, A: Quantitative reverse transcription-PCR with Tim17A-specific primers, the results are standardized by the control gene GAPDH, 16N in the figure, HME is normal mammary epithelial cells, and the rest are breast cancer cells; B: breast cancer cells Immunohistochemical detection of Tim17 protein, cell markers such as A; C: Immunohistochemical detection of Tim17 protein in tissue, the picture shows examples of normal, carcinoma in situ, and invasive carcinoma; top: normal; middle: ductal carcinoma in situ; Bottom: invasive carcinoma.

Figure 2 Differences in the expression of TIMM17A RNA between normal controls and breast cancer patients (data mining results), among them, Class 1: Normal (7 cases); Class 2: Breast Carcinoma (45 cases).

Figure 3 The expression level of TIMM17A is significantly positively correlated with the prognosis of breast cancer patients (data mining results),

Among them, A: Dataset 1, Class 1: Survival (121 cases), Class 2: Death (38 cases); B: Dataset 2, Class 1: No disease (180 cases), Class 2: Relapse (93 cases) .

Figure 4 The expression level of TIMM17A is significantly positively correlated with the grade and degree of differentiation of breast cancer (data mining results),

Among them, A: data set 1, Class 1: grade 1 (68 cases), Class 2: grade 2 (126 cases), Class 3: grade 3 (55 cases); B: data set 2, Class 1: grade 1 ( 67 cases), Class 2: grade 2 (128 cases), Class 3: grade 3 (54 cases); C: data set 3, Class 1: well differentiated (30 cases), Class 2: moderately differentiated (83 cases), Class 3: Poorly differentiated (83 cases).

Figure 5 TIMM17A is overexpressed in many tumors and is a broad-spectrum marker (Oncomine data analysis results). Among them, A: bladder cancer; B: bowel cancer; C: liver cancer; D: lung cancer; E: prostate cancer.

Detailed ways

In the first aspect of the present invention, a novel isolated differentially expressed Tim17 polypeptide is provided, which comprises: a polypeptide having an amino acid sequence of SEQ ID NO: 1, or a conservative variant polypeptide thereof, or an active polypeptide thereof, or an active polypeptide thereof derivative.

Example 1: Obtaining differentially expressed Tim17 polypeptides

Breast cancer cell lines isolated from a patient, including normal (16N), tumor (NT), and metastases (MT2), were cultured with stable isotope D3-Leu (Cambridge Isotope) to label tumor cells. Normal and metastatic tumor cells were cultured with D0-Leu-containing medium. The proteins of the three cell lines were extracted with lysate (8M urea, 2.5Mthiourea, 65mMDTT, 4% (w/v) CHAPS, 0.5% (v/v) Biolytes pH 3-10, protease inhibitor cocktail) and quantified (RC DC Protein Assay Kit, Biorad company). Equal amounts of proteins were pooled into a normal:tumor group and a tumor:metastatic group, and then separated by one-dimensional SDS gel electrophoresis. After the gel was stained, each gel strip was cut into 8 bands of similar size. Each band undergoes decolorization, dry gel, in-gel enzyme digestion, peptide extraction and other steps, and then separates the peptide by liquid chromatography. The isolated polypeptides were identified by LTQ-Orbitrap (ThermoElectron) mass spectrometer. Quantitative information was obtained for more than 1200 proteins identified. More than 70 of them were identified as significantly differentially expressed proteins. Excluding known tumor markers, among the remaining new markers, it was determined that differentially expressed significantly TIMM17A (one of two isoforms of Tim17 protein), which is a protein with important mitochondrial functions. The peptide for which quantitative information was obtained is GKEDPWNSITSGALTGAILAAR. In tumors, its expression is 4.8 times higher than in normal cells. The same results were obtained through multiple mass spectrometry identifications.

In a second aspect of the invention there is provided a polynucleotide encoding the isolated polypeptide. SEQ ID NO: 4.

Example 2: Obtaining the full-length gene encoding TIMM17A from a breast cancer cell line

Total RNA in 21T breast cancer cell lines (derived from NT or MT2 cells) was extracted with Trizol reagent (Invitrogen), and the total RNA was reverse-transcribed into cDNA with PrimeScript lst strand cDNA Synthesis Kit (TaKaRa). Using the synthesized cDNA as a template, design the TIMM17A clone gene forward primer (the original TIMM17A start codon ATG has been removed) 5'-CA GAA TTC TGG AGG AGT ACGCGC GAG-3' and reverse primer containing restriction sites 5'-GACCTCGAGCTACTGATATTGTCGATA-3', use hot-start Taq DNA polymerase (TaKaRa company) for PCR reaction, the reaction conditions are 98 degrees pre-denaturation for 2 minutes, then 95 degrees for 10 seconds, 55 degrees for 15 seconds, 72 degrees for 40 seconds Extend the reaction to reflect 30 cycles. The PCR product and the pcDNA3.O-HA-FLAG empty vector were subjected to EcoRI and XhoI double enzyme digestion, ligation, and DH5α bacterial transformation experiments to obtain positive clones transformed by the TIMM17A gene. After DNA sequencing confirmed that the gene sequence was correct, pcDNA3.O was obtained - HA-FLAG-TIMM-17A vector.

In the third aspect of the present invention, compounds that mimic, promote and antagonize the activity of human Tim17, and compounds that inhibit the expression of human Tim17 are provided. Preferably, the compound is a small molecule interference RNA of human Tim17 coding sequence or a fragment thereof. The small molecule shRNA was cloned into the pLVTHM vector to form pLVTHM-sTimm17A. The vector and the packaging plasmid were co-transfected into the packaging cell 293T to produce the virus. Infect the tumor cells to be tested with the virus, and find that the growth of the tumor cells to be tested is obviously slowed down.

Example 3: TIMM17A small molecule interfering RNA inhibits tumor growth

According to the shRNA design rules, three sequences of 23 base shRNAs were designed. The sequences which have a better effect of inhibiting gene expression were cloned into the MluI and ClaI sites of the pLVTHM (Trono Laboratory, Switzerland) vector. The resulting vector was named pLVTHM-sTimm17A. To generate inducible shRNA-expressing cell lines, pLVPT-tTR/KRAB-Red vector (Trono Laboratories, Switzerland) was transformed into 293T cells. At the same time, two packaging vectors PMD.G (VSV.G), CMV-DR891 were co-transfected into 293T cells. After the transfected 293T cells continued to grow for 48 hours, the supernatant of the cells was collected. Cells were removed by filtration, and the supernatant was infected with MDA-231 breast cancer cells. After the infected MDA-231 cells were cultured for 48 hours, they were sorted by flow cytometry to obtain cells expressing KRAB inhibitors with red fluorescence. The MDA-231-KRAB cell line stably expressing KRAB was obtained. The constructed pLVTHM-sTimm17A vector, PMD.G (VSV.G), and CMV-DR891 packaging vector were cotransfected into 293T cells to produce shRNA expression vector virus particles. The MDA-231-KRAB cell line was infected with the virus in the same way as above to generate the stable cell line, thereby producing the MDA-231-KRAB-sTimm17A cell line that can be induced to express shRNA by DOX (Sigma Company). The cells express shRNA under 1 μg/ml DOX to inhibit the expression of TIMM17A gene.

On this basis, the MDA-231-KRAB-sTimm17A cell line was seeded in 10 wells of a 96-well cell culture dish at a density of 50,000/well. Among them, 5 wells were cultured in DOX-containing medium, and the other 5 wells were cultured in DOX-free medium. After 72 hours, the cell growth was measured by the MTT (Sigma company) method. The results showed that the growth of MDA-231-KRAB-sTimm17A cells cultured in DOX medium was inhibited by 50% compared with the cells without DOX.

In the fourth aspect of the present invention, vectors and viruses containing the above-mentioned small interfering RNA are provided. The vector and virus production process has been detailed in Example 3.

In a fifth aspect of the present invention, a method for detecting the presence of Tim17 in a sample is provided. It includes: extracting RNA from tissue and reverse transcribing it into cDNA. Quantitative PCR amplification was carried out with the designed and synthesized primer sequence (SEQ ID NO: 3). Another way to detect the protein level is to extract the protein in the tissue and perform immunoblotting reaction with specific antibody. Alternatively, perform immunohistochemistry with specific antibodies on paraffin-embedded tissue. Also provided is a method for detecting tumors, including: performing the above-mentioned quantitative PCR test, western blot test or immunohistochemical test on samples, and the individual with a result higher than the background level has a tumor or the susceptibility of the tumor is higher than that of the normal population.

Embodiment 4: Quantitative PCR detects the RNA of TIMM17A

According to the corresponding protein sequence identified by mass spectrometry and the gene sequence information on the NCBI website (accession number: NM_006335.1), TIMM17A primers were designed using primer3 software (http://frodo.wi.mit.edu/primer3/input.htm) . The obtained upstream primer sequence is atccctggaactccatcaca from 5' to 3', and the downstream primer sequence is tgcagaggcaaatcttgtca. The primers were designed so that the amplified product spanned the No. 2 intron of the genome sequence, thereby reducing the possibility of genomic DNA affecting the results. The internal reference of quantitative PCR was GAPDH gene. The upstream primer of the GAPDH gene used was cgagatccctccaaaatcaa, and the downstream primer was ttcacacccatgacgaacat. Total RNA was extracted with Trizol, and the concentration and purity of RNA were measured by a spectrophotometer (260nm/280nm). 3 μg of total RNA and oligo dT primers were used for reverse transcription (TaKaRa) by PrimeScript lst strand cDNASynthesis Kit. In the 25 μl quantitative PCR reaction system, add 75ng cDNA template, 1×SYBR Premix Ex Taq (Perfect Real Time; TaKaRa) and each 200nM primer. The amplification reaction was carried out with iQ5 quantitative PCR instrument (Bio-rad Company). The reaction program was: initial denaturation at 95°C for 10 seconds, 40 cycles of two-step method, denaturation at 95°C for 5 seconds, annealing at 60°C, and extension for 20 seconds. Quantitative data analysis was performed in relative quantitative mode with iQ5 (Bio-Rad) v2.0 software. Figure 1A is a result of a quantitative PCR experiment. Using cell line RNA, it was found that normal cells 16N and HME expressed very low TIMM17A RNA, while tumor cells such as NT, MT2, and T47D expressed significantly higher levels than normal cells.

Example 5: Western blot and immunohistochemical detection of Tim17 protein

Antibody to Tim17 (rabbit anti-human TIMM17A/TIMM17B antibody, PTGLAB company) was used to perform western blot and immunohistochemical experiments in cells and tissues. In immunoblotting, approximately 30 μg of total protein was separated on a 6-15% gradient SDS-PAGE gel and transferred to a PVDF membrane (Amersham Bioscience). Block the PVDF membrane with 5% BSA-containing TBST buffer (137mM NaCl, 20mM Tris-HCl pH7.6, 0.1% Tween 20), incubate with Tim17 antibody for one hour at room temperature or overnight at 4°C, wash, and HRP-coupled Anti-incubation. The washed membrane was developed with ultra-sensitive chemiluminescence solution (Amersham Bioscience), and imaged by LAS-3000 imaging system (Fuji, Japan). The bands of western blot were quantified by Multi Gauge V3.0 (Fuji, Japan) software. Figure 1B is the result of Western blot experiments in cell lines. Actin B antibody (mouse anti-human Actin B antibody, PTGLAB company) was used as a control for loading samples. Experiments showed that in normal breast epithelial cells, 16N and HME, Tim17 was expressed at a very low level, while in breast cancer cells, NT and MT2, the expression level of Tim17 was significantly increased.

In immunohistochemical experiments, formalin-fixed and paraffin-embedded tissue chips were purchased from US Biomax (CA, U.S.A. Catalog: TMA-BR480). After the sections were dewaxed and hydrated, they were boiled in 0.01M citrate buffer (pH=6.0) to restore the antigen. Incubate overnight for 4 hours with primary antibody against Tim17 diluted 1:100. It was then detected by the anti-rabbit HRP polymer detection system and imaged by the Olympus BX51 microscope system. Stained tissue microarrays were scored by two investigators, and microarrays in which at least 10% of breast epithelial cells were stained were counted as positive microarrays. Statistical significance test was carried out with Fisher Exact method. P<0.05. Figure 1C illustrates immunohistochemical staining of normal, ductal carcinoma in situ, and invasive carcinoma. The results showed that Tim17 protein was not expressed in all five normal tissues, and Tim17 protein was first expressed in part in early breast disease and mammary gland hyperplasia; the expression peak was reached in early tumor in situ ductal carcinoma (100% of the detected tumors Express, 6/6). The results showed that Tim17 can be used as a marker for breast cancer prevention and early detection.

In the sixth aspect of the present invention, uses of the polypeptides and coding sequences of the present invention are provided. The application includes immunizing animals with the polypeptide to obtain antibodies against the polypeptide, and then detecting the expression of the protein through the antibody. It can be detected by blood, tissue, and other bodily fluids (such as breast aspirate, nipple aspirate). The role of the coding sequence includes the design of primers, probes, and siRNA, shRNA, etc. to detect the expression and mutation of DNA and RNA, and to achieve the therapeutic effect of inhibiting tumor growth due to the inhibition of gene expression. The use is described in Examples 3, 4, and 5.

In the seventh aspect of the present invention, a pharmaceutical combination is provided, which contains a safe and effective amount of the human Tim17 antagonist of the present invention and a pharmaceutically acceptable carrier. Preferred antagonists are small interfering RNA sequences. These pharmaceutical compositions can treat tumors. Specifically, pLVTHM-sTimm17A constructed with the pLVTHM vector, and the defective virus produced therefrom. These results are described in Example 3.

Other aspects of the invention will be apparent to those skilled in the art from the technical disclosure herein.

Table 1 is the expression of Tim17 in human normal and tumor tissues.

Table 1

Among them, ^a Lobular carcinoma in situ; ^b Ductal carcinoma in situ;

^c Infiltrating carcinoma; ^d Age; P value calculated by Fisher Exact statistics. .

Claims (4)

1. The sequence is shown as SEQ ID NO: 1 in the preparation of a mass spectrum or antibody-based tumor marker detection or diagnostic preparation.

A Tim17 polypeptide encoding protein sequence SEQ ID NO: 2 in preparing a preparation for inhibiting tumor growth, wherein the inhibition mode is that a synthetic compound pLVTHM-sTimm17A containing small molecule interference RNA of a human Tim17 coding sequence or a fragment thereof is used for inhibiting the TIMM 17A;

the pLVTHM-sTimm17A is a 23-base shRNA with three sequences designed according to shRNA design rules, wherein the sequences with better inhibition gene expression effects are cloned to MluI and ClaI sites of a pLVTHM vector, and the generated vector is pLVTHM-sTimm17A.

3. Use according to claim 1 or 2, wherein the tumour is breast cancer, lung cancer, liver cancer, bowel cancer, bladder cancer, prostate cancer or lymphoma.

4. Use according to claim 1 or 2, wherein said neoplasm is breast cancer.

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