CN104232744B - Application of human CIT gene and EGFR gene in curing tumors and related drugs - Google Patents

Abstract

The invention relates to the field of biotechnology, and specifically discloses a single human CIT gene; and the use of the human CIT gene and the human EGFR gene as synergistic drug delivery targets in the preparation or screening of tumor treatment drugs. The present invention further constructs CIT gene small interfering RNA, CIT gene interference lentivirus vector, and CIT gene interference lentivirus, and discloses their use in synergistic treatment of tumors with EGFR gene. The siRNA provided by the present invention or the lentiviral vector or lentivirus containing the siRNA sequence can specifically inhibit the expression of human CIT gene and/or human EGFR gene, especially the lentivirus, and the CIT gene interference lentivirus of the present invention can be alone, or Synergistically inhibiting tumor cell growth and promoting tumor cell apoptosis with EGFR gene interference lentivirus is of great significance in tumor therapy.

Description

Application of human CIT gene and EGFR gene in tumor therapy and related drugs

technical field

The invention relates to the field of biotechnology, in particular to a single human CIT gene; and the use of human CIT gene and human EGFR gene in synergistic treatment of tumors and related medicines.

Background technique

At present, the gene therapy of tumor has achieved vigorous and rapid development, but so far, there are still many problems to be solved. At present, there are too few genes used in tumor gene therapy, and there are only a few genes that can inhibit tumor growth, so it is urgent to provide more available genes. In addition, since the process of tumor occurrence, development, and metastasis is a complex network system involving multiple genes and involving multiple signaling pathways, the efficacy of single gene therapy or single therapy is often limited. Therefore, the focus of future gene therapy development will be to mine and identify clinically important genes, to explore the combination therapy of multiple genes with different anti-tumor mechanisms, and to combine multi-gene targeted drug therapy.

CIT citron (Rho-interacting, serine/threonine kinase 21, serine/threonine kinase 21 interacting with Rho) contains a protein kinase domain similar to ROCK (Rho-associated kinase, Rho-connected kinase). The body, called Citron-K, is the first known downstream target of Rho-GTP and is characterized by cell cycle-dependent expression. Citron-K accumulates in interphase cells, disperses in the cytoplasm in the prometaphase of mitosis, distributes in the cell cortex in the anaphase, and accumulates in the cleavage groove at the end stage (Madaule P, Eda M, Watanabe N, et al.Role of citronkinase as a target of the small GTPase rho in cytokinesis.Nature.1998;394(6692):491-494.Paramasivam M,Chang YJ,Lotrco JJ.ASPM and citron kinase co-localize to midboby ring during cytokinesis.Cell Cycle.2007;6(13 ) 105-1612). Citron kinase, a rho-dependent kinase, induces di-phosphorylation of regulatory light chain of myosin by regulating myosin contraction and protoplasmic movement during cytoplasmic division II. Mol Biol Cell. 2003; 14(5):1745-1756.). A variant that overexpresses the citron gene results in multinucleated cells, and a variant that loses kinase activity exhibits abnormal shrinkage during cytoplasmic migration.

Studies have reported that the citron gene plays a functional role in some physiological processes of neuron formation and spermatogenesis (Di Cunto F, Imarisio S, Hirsch E, et al. Defective Neurogenesis in CitronKinase Knockout Mice by Altered Cytokinesis and Massive Apoptosis .Neuron2000;28(1):115-127.Di Cunto F,Imarisio S,Camera P,et al.Essential role of citron kinase in cytokinesis of spermatogenic precursors.J Cell Sci.2002;115(Pt 24):4819-4826 .), mice knocked out of the citron gene were deformed or died within a few weeks of birth due to ataxia or epilepsy (Di Cunto F, Imarisio S, Hirsch E, et al. Defective Neurogenesis in Citron Kinase Knockout Mice by Altered Cytokinesis and Massive Apoptosis. Neuron 2000;28(1):115–127.). At the same time, Citron-K is necessary for the physiological expansion of undifferentiated male gametocyte precursors and ensures the transformation of spermatogonia into spermatocytes. Citron-knockout male mice exhibited double testis-like lesions, while the ovaries of female mice were not Morphological changes occur (Di Cunto F, Imarisio S, Camera P, et al. Essential role of citron kinase in cytokinesis of spermatogenic precursors. J Cell Sci. 2002; 115(Pt24): 4819-4826.). And there are research reports that Citron gene is also involved in the replication process of virus (Atreya CD, Kulkarni S, Mohan KV.Rubella virus P90 associates with the cytokinesis regulatory protein citron-K kinase and theviral infection and constitutive expression of P90protein both induce cellcycle arrest following S phase in cell culture. Arch Virol. 2004; 149(4):779-789. Atreya CD, Mohan KV, Kulkarni S. Rubella virus and birth defects: molecular insights into the viral teratogenesis at the cellular level. Birth Defects ResA Clin Mol Teratol. 2004; 70(7): 431-437. Loomis RJ, Holmes DA, Elms A et al. Citronkinase, a rho A effector, enhances HIV-1 virion production by modulating exocytosis. Traffic. 2006; 7(12): 1643-1653. ). Therefore, citron gene plays an important role in a series of important physiological processes. Citron gene depends on cell cycle expression and plays an important physiological function in cell mitosis. Cancer tissue is the continuous proliferation of cells to form immortality. Therefore, citron gene It may have a certain relationship with the occurrence of tumors, and it is expected to become a new target for tumor gene therapy.

Epidermal growth factor receptor (EGFR) is a transmembrane protein belonging to the ErbB receptor family, and its ligands (EGF, TGF-α, etc.) bind to the extracellular segment of EGFR to dimerize it. This leads to the activation of intracellular tyrosine kinase and a series of signal transduction cascade reactions, promoting cell proliferation, angiogenesis, metastasis and inhibiting cell apoptosis (Liang Houjie, Zou Lan. EGFR targeted drug therapy for advanced colorectal cancer Recent Advances. China Prescription Drugs 2009;84:58-61.), thus leading to tumorigenesis. A large number of studies have found that the EGFR gene is overexpressed or abnormally activated in a variety of tumor tissues, thereby enhancing the proliferation, invasion and metastasis of tumor cells. EGFR has become a clinically validated therapeutic target for various types of tumors (Ciardiello F, Tortora G. EGFR antagonists in cancer treatment. N Engl J Med 2008; 358:1160-1174.).

RNAi is a sequence-specific post-transcriptional gene silencing phenomenon mediated by double-stranded RNA. It has become an emerging and effective method for studying gene function, and is expected to become a tool for gene therapy of tumors and other diseases (Izquierdo M.Shortinterfering RNAs as a tool for cancer gene therapy. Cancer Gene Ther. 2005; 12(3):217-27.). Lentivirus (lentivirus) vector is an efficient gene transduction tool, mainly used to infect cells with low transfection efficiency by conventional methods, such as primary cells. Lentiviral particles can infect proliferating and non-proliferating cells at the same time, and the infection is relatively stable and long-lasting. The use of lentiviral vectors can achieve high expression of specific genes, and can also express hairpin RNA (short hairpin RNA, shRNA) to down-regulate the expression of a gene in the form of RNA interference (RNAi). The present invention intends to use lentivirus-mediated RNAi technology to study the function of CIT gene in synergizing with EGFR gene in inhibiting tumor cell proliferation, so as to provide a theoretical basis for non-surgical clinical treatment of malignant tumors.

Contents of the invention

The purpose of the present invention is to disclose the CIT gene as the target of cancer treatment, the purposes of treating tumors and related medicines; Use of synergistically treating tumors, and tumor therapeutic drugs thereof.

The present invention uses RNA interference as a means to study the role of a single CIT gene in the occurrence and development of tumors, and the synergistic effect of CIT gene and EGFR gene in the occurrence and development of tumors, and discloses a method for inhibiting or reducing the growth of tumor cells. , a method for proliferation, differentiation and/or survival, the method comprising: administering to tumor cells a drug that can specifically inhibit the transcription and translation of CIT gene and/or EGFR gene, or that can specifically inhibit the expression of CIT protein and/or EGFR protein Active substances to inhibit the growth, proliferation, differentiation or survival of tumor cells.

The first aspect of the present invention discloses the use of the isolated human CIT gene in the preparation or screening of drugs for treating tumors, or in the preparation of drugs for diagnosing tumors.

The tumor can be any tumor whose tumor cell proliferation is related to the expression of human CIT gene; further, it is a malignant tumor, for example selected from: colon cancer.

In the present invention, the human CIT gene is used as a target for tumor cells in the preparation or screening of tumor therapeutic drugs, or in the preparation of tumor diagnostic drugs. Further, the target for tumor cells is an RNA interference target.

The use of the isolated human CIT gene for the preparation or screening of tumor therapeutic drugs includes two aspects: first, the human CIT gene is used as a target of the drug or preparation against tumor cells in the preparation of tumor therapeutic drugs; second, the The human CIT gene is used as a target of drugs or preparations for tumor cells to screen tumor therapeutic drugs.

The application of the CIT gene as the target of drugs or preparations for tumor cells in the preparation of tumor therapeutic drugs specifically refers to: using the CIT gene as the target of RNA interference to develop drugs or preparations for tumor cells, thereby increasing or reducing tumor Expression levels of the CIT gene in cells.

The application of the CIT gene as the target of drugs or preparations for tumor cells in the screening of tumor therapeutic drugs specifically refers to: using the CIT gene as the target of action, screening the drugs or preparations to find the ones that can inhibit or promote the expression of human CIT genes. Drugs as candidates for cancer treatment. The CIT gene small molecule interfering RNA (siRNA) according to the present invention is obtained by screening the human CIT gene as an action object, and can be used as a drug capable of inhibiting tumor cell proliferation. In addition, such as antibody drugs, small molecule drugs, etc., can also target the CIT gene and its protein.

The use of the CIT gene in the preparation of tumor diagnostic drugs refers to the use of CIT gene expression products as a tumor diagnostic index in the preparation of tumor diagnostic drugs.

The tumor treatment drug is a molecule that can specifically inhibit the transcription or translation of the CIT gene, or can specifically inhibit the expression or activity of the CIT protein, thereby reducing the expression level of the CIT gene in tumor cells, and inhibiting the proliferation and growth of tumor cells. , differentiation and/or survival purposes.

The first aspect of the present invention also discloses the use of isolated human CIT gene and human EGFR gene in the preparation or screening of tumor therapeutic drugs, or in the preparation of tumor diagnostic drugs.

In the present invention, the human CIT gene and human EGFR gene are used as targets for tumor cells in the preparation or screening of tumor therapeutic drugs, or in the preparation of tumor diagnostic drugs. Further, the target for tumor cells is an RNA interference target.

The use of the isolated human CIT gene and human EGFR gene for the preparation or screening of tumor therapeutic drugs includes two aspects: first, the human CIT gene and human EGFR gene are used as targets of drugs or preparations for tumor cells in the preparation of Drugs for treating tumors; secondly, using human CIT gene and human EGFR gene as the targets of drugs or preparations for tumor cells to screen for drugs for treating tumors.

The application of human CIT gene and human EGFR gene as targets of drugs or preparations for tumor cells in the preparation of tumor therapeutic drugs specifically refers to: using CIT gene and human EGFR gene together as targets of RNA interference, and developing drugs that can simultaneously target both. Tumor therapy drugs or preparations for these genes, thereby increasing or reducing the expression levels of CIT gene and human EGFR gene in tumor cells; or, using CIT gene and human EGFR gene as the targets of RNA interference respectively, to develop drugs targeting the two genes respectively The tumor treatment drug, so as to obtain the tumor treatment drug or preparation that can increase or decrease the expression level of CIT gene and human EGFR gene in tumor cells.

The said application of human CIT gene and human EGFR gene as targets of drugs or preparations for tumor cells to screen tumor therapeutic drugs specifically refers to: using human CIT gene and human EGFR gene as action objects at the same time to screen drugs to find A drug that can affect (inhibit or promote) human CIT gene expression and a drug that can affect (inhibit or promote) human EGFR gene expression respectively, and then use it as a candidate composition drug for tumor treatment; or find a drug that can simultaneously affect (inhibit or promote) human CIT Genes and drugs expressed by human EGFR genes are candidates for tumor treatment. CIT gene small molecule interfering RNA (siRNA), gene interference nucleic acid construct, lentivirus as described in the present invention, and EGFR gene small molecule interference RNA (siRNA), gene interference nucleic acid construct, lentivirus are all based on CIT gene and EGFR gene are respectively screened for the target of action; when they are used together, there is a synergistic effect, which is better than that of a single substance; it can be used as a drug with the effect of inhibiting tumor cell proliferation. In addition, such as antibody drugs, small molecule drugs, etc., can also use human CIT gene and human EGFR gene and their proteins as the targets of action.

The said use of human CIT gene and human EGFR gene for the preparation of tumor diagnostic drugs refers to the use of CIT gene expression products and human EGFR gene expression products as tumor diagnostic indicators in the preparation of tumor diagnostic reagents.

The tumor treatment drug of the present invention is a molecule that can specifically inhibit the transcription or translation of human CIT gene and/or human EGFR gene, or can specifically inhibit the expression or activity of human CIT gene and/or human EGFR gene, thereby reducing The expression level of human CIT gene and/or human EGFR gene in tumor cells can achieve the purpose of inhibiting the proliferation, growth, differentiation and survival of tumor cells.

The tumor treatment drugs prepared or screened by the present invention include but not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs (such as inhibitors), antibody drugs, polypeptides, proteins or interfering lentiviruses.

The nucleic acid molecules include, but are not limited to: antisense oligonucleotides, double-stranded RNA (dsRNA), ribozyme, small interfering RNA (esiRNA) or short hairpin RNA (shRNA) prepared by endoribonuclease III.

The administration amount of the tumor treatment drug is sufficient to reduce the transcription or translation of human CIT gene and human EGFR gene, or the dosage sufficient to reduce the expression or activity of human CIT protein and human EGFR protein. Such that the expression of human CIT gene and human EGFR gene is reduced by at least 50%, 80%, 90%, 95% or 99%.

In the present invention, when the human CIT gene and the human EGFR gene are used as the targets of synergistic drug administration for tumor treatment and drug screening, the method of RNA interference is used, and the human CIT gene and the EGFR gene are used as the target genes of RAN interference to reduce the two gene expression level.

The tumor can be any tumor whose tumor cell proliferation is related to the expression of human CIT gene and human EGFR gene; further, it is a malignant tumor, for example selected from: colon cancer.

The method of using the above-mentioned tumor therapeutic drugs to treat tumors is to suppress the proliferation of tumor cells by reducing the expression level of human CIT gene alone to achieve the purpose of treatment, or to inhibit the proliferation of tumor cells by simultaneously reducing the expression levels of human CIT gene and human EGFR gene. achieve the purpose of treatment. Specifically, during treatment, the substance that can effectively reduce the expression level of human CIT gene; or, the substance that can effectively reduce the expression level of human CIT gene and human EGFR gene is administered to the patient.

The second aspect of the present invention discloses a nucleic acid molecule drug for treating tumors, the active ingredient of which contains an isolated nucleic acid molecule that reduces the expression of the CIT gene in tumor cells, and an isolated nucleic acid molecule that reduces the expression of the EGFR gene in tumor cells; The isolated nucleic acid molecule expressed by the CIT gene in a tumor cell comprises:

a) CIT gene double-stranded RNA, which contains a nucleotide sequence capable of hybridizing with the CIT gene under stringent conditions; or

b) CIT gene shRNA, which contains a nucleotide sequence capable of hybridizing with the CIT gene under stringent conditions;

The isolated nucleic acid molecule that reduces EGFR gene expression in tumor cells comprises:

A. EGFR gene double-stranded RNA, which contains a nucleotide sequence capable of hybridizing with the EGFR gene under stringent conditions; or

B. EGFR gene shRNA, the EGFR gene shRNA contains a nucleotide sequence capable of hybridizing with the EGFR gene under stringent conditions.

Further, the tumor is colon cancer.

Preferably, the double-stranded RNA of the CIT gene comprises a first strand and a second strand, the first strand and the second strand are complementary to form an RNA dimer, and the sequence of the first strand is identical to that of the CIT gene The target sequences are basically the same.

Preferably, the EGFR gene double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary to form an RNA dimer, and the sequence of the first strand is identical to that of the EGFR gene The target sequences are basically the same.

Preferably, the CIT gene shRNA includes a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, and the sequences of the sense strand segment and the antisense strand segment Complementary, and the sequence of the sense strand fragment is basically the same as the target sequence of the CIT gene.

Preferably, the EGFR gene shRNA includes a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, and the sequences of the sense strand segment and the antisense strand segment complementary, and the sequence of the sense strand fragment is basically identical to the target sequence of the EGFR gene.

More preferably, the sequence of the stem-loop structure of the shRNA can be selected from any of the following: UUCAAGAGA, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU and CCACACC.

More preferably, the target sequence of the CIT gene is the sequence shown in SEQ ID NO:1; the target sequence of the EGFR gene is the sequence shown in SEQ ID NO:2.

The target sequence of the CIT gene is the fragment in the CIT gene corresponding to the mRNA fragment complementary to the siRNA when the siRNA is used to specifically silence the expression of the CIT gene. Similarly, the target sequence of the EGFR gene is the fragment in the EGFR gene corresponding to the mRNA fragment complementary to the siRNA when the siRNA is used to specifically silence the expression of the EGFR gene.

The length of the first strand and the second strand of the double-stranded RNA is 15-27 nucleotides; preferably, the length is 19-23 nucleotides; the best, the length is 19, 20 or 21 nucleotides.

Further, the double-stranded RNA is small interfering RNA (siRNA).

Optimally, the sequence of the first strand of the small interfering RNA of the CIT gene is shown in SEQ ID NO: 9, specifically 5'-GUCCUCAUACCAGGAUAAA-3'. The first strand of the CIT gene siRNA shown in SEQ ID NO: 9 is a strand in the small interfering RNA designed for the human CIT gene with the sequence shown in SEQ ID NO: 1 as the RNA interference target sequence, the first strand The siRNA composed of the second strand can specifically silence the expression of endogenous CIT gene in tumor cells.

Optimally, the sequence of the first strand of the small interfering RNA of the EGFR gene is shown in SEQ ID NO: 10, specifically 5'-GGCUGGUUAUAUGUCCUCAUU-3'. The first strand of the EGFR gene siRNA shown in SEQ ID NO: 10 is a strand in the small interfering RNA against the human EGFR gene designed with the sequence shown in SEQ ID NO: 2 as the RNA interference target sequence, the first strand The siRNA combined with the second strand can specifically silence the expression of endogenous EGFR gene in tumor cells.

Optimally, the sequence of the CIT gene shRNA is shown in SEQ ID NO: 11, specifically: 5'-GCGUCCUCAUACCAGGAUAAACUCGAG UUUAUCCUGGUAUGAGGACGC-3'.

Optimally, the sequence of the EGFR gene shRNA is shown in SEQ ID NO: 12, specifically: 5'-GUGGCUGGUUAUGUCCUCAUUCUCGAGAAUGAGGACAUAACCAGCCAC-3'.

When used as a drug for treating tumors, a safe and effective amount of double-stranded RNA or shRNA is administered to mammals. The specific dosage should also consider factors such as the route of administration, the health status of the patient, etc., all of which are within the skill of skilled physicians.

The third aspect of the present invention discloses a lentiviral vector drug for treating tumors, the active ingredient of which contains a CIT gene interference lentiviral vector and an EGFR gene interference lentiviral vector, and the CIT gene interference lentiviral vector contains shRNA encoding the aforementioned CIT gene The gene fragment of the EGFR gene interference lentiviral vector contains the gene fragment encoding the aforementioned EGFR gene shRNA.

Further, the tumor is colon cancer.

The CIT gene interference lentiviral vector is obtained by cloning the DNA fragment encoding the aforementioned CIT gene shRNA into a known carrier, and most of the known vectors are lentiviral vectors, and the CIT gene interference lentiviral vector is packaged into an infectious After the strong virus particles are infected, the tumor cells are then transcribed, and the shRNA is transcribed, and the small interfering RNA of the CIT gene is finally obtained through steps such as enzyme digestion and processing, which are used to specifically silence the expression of the CIT gene. The EGFR gene interference lentiviral vector is the same as the CIT gene.

The DNA sequence encoding the shRNA gene fragment of the CIT gene contains the sequence shown in SEQ ID NO: 1 and its complementary sequence. The DNA sequence encoding the EGFR gene shRNA gene fragment contains the sequence shown in SEQ ID NO: 2 and its complementary sequence.

Further, the gene interference lentiviral vector of the present invention also contains a promoter sequence and/or a nucleotide sequence encoding a detectable marker in tumor cells; preferably, the detectable marker such as green Fluorescent protein (GFP).

Further, the lentiviral vector can be selected from: pLKO.1-puro, pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagRFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti6-GW/U6-laminshrna, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or pLenti6.2/N-Lumio/ Either of V5-GW/lacZ.

In the fourth aspect of the present invention, a lentiviral drug for treating tumors is disclosed, the active ingredient of which contains CIT gene interference lentivirus and EGFR gene interference lentivirus. With the assistance of virus packaging plasmids and cell lines, the EGFR gene interference lentivirus is packaged by viruses with the assistance of the aforementioned EGFR gene interference lentivirus vectors with the assistance of lentivirus packaging plasmids and cell lines.

Further, the tumor is colon cancer.

The lentivirus of the present invention can infect tumor cells and produce small molecular interference RNAs respectively targeting CIT gene or EGFR gene, thereby inhibiting the proliferation of tumor cells. The CIT gene interference lentivirus and the EGFR gene interference lentivirus can be used to prepare drugs for preventing or treating tumors.

In the fifth aspect of the present invention, a pharmaceutical composition for treating colon cancer is disclosed, which comprises the aforementioned isolated nucleic acid molecule for reducing the expression of CIT gene in tumor cells, a CIT gene interference lentiviral vector, and a CIT gene interference lentiviral medium. and at least one of the aforementioned isolated nucleic acid molecules that reduce EGFR gene expression in tumor cells, EGFR gene interference lentiviral vectors, and EGFR gene interference lentiviruses.

Preferably, the above ingredients are the active ingredients of the pharmaceutical composition for treating colon cancer.

When used as a drug for treating tumors, a safe and effective amount of double-stranded RNA, shRNA, or lentivirus is administered to mammals. The specific dosage should also consider factors such as the route of administration, the health status of the patient, etc., all of which are within the skill of skilled physicians.

In the pharmaceutical composition of the present invention, the active ingredients that specifically silence the expression of the CIT gene and the expression of the EGFR gene play a role in synergistic therapy; as described in the examples of the present invention, the growth inhibition rate of cancer cells after double gene knockout is obvious It is greater than the sum of the single gene knockdown group, indicating that the substance that specifically silences the expression of the CIT gene and the substance that specifically silences the expression of the EGFR gene cooperate to inhibit the proliferation of tumor cells.

Further, the medicine or pharmaceutical composition of the present invention contains 1-99wt% of the small interfering RNA, shRNA, gene interference nucleic acid construct and/or gene interference lentivirus, and a pharmaceutically acceptable carrier, diluent or excipient Forming agent.

When preparing a drug, the active ingredient is usually mixed with an excipient, or diluted with an excipient, or enclosed in a carrier that can exist in the form of a capsule or a sachet. When the excipient acts as a diluent, it can be a solid, semi-solid or liquid material which acts as a vehicle, carrier or medium for the active ingredient. Thus, the composition can be in the form of tablets, pills, powders, solutions, syrups, sterile injectable solutions, and the like. Examples of suitable excipients include: lactose, dextrose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, and the like. The formulation may also include: wetting agents, emulsifiers, preservatives (such as methyl and propylparaben), sweeteners, and the like.

The beneficial effects of the present invention are: the siRNA provided by the present invention or the nucleic acid construct comprising the small interfering RNA sequence, and the lentivirus can specifically inhibit the expression of the human CIT gene, especially the lentivirus, which can be used alone or with EGFR-targeted lentivirus Viruses synergistically inhibit tumor cell growth and promote tumor cell apoptosis, which is of great significance in tumor therapy. The CIT gene in the present invention can be used not only as a single tumor treatment target, but also as an EGFR gene synergistic treatment target, which is of great significance in tumor treatment.

Description of drawings

Figure 1: GV115 plasmid DNA map

Figure 2: GV113 plasmid DNA map

Figure 3: CIT shRNA plasmid identification map

1#: Negative control (ddH2O); 2#: Negative control (empty self-connected control group)

3#: Marker from top to bottom are 5kb, 3kb, 2kb, 1.5kb, 1Kb, 750bp, 500bp, 250bp, 100bp

4#～8#: Identification of CIT shRNA 1-5 transformants

Figure 4: EGFR shRNA plasmid identification map

1#: negative control (ddH2O)

2#: Negative control (no-load self-connection control group)

3#: Marker from top to bottom are 5kb, 3kb, 2kb, 1.5kb, 1Kb, 750bp, 500bp, 250bp, 100bp

4#～8#: Identification of EGFR shRNA 1-5 transformants

Figure 5: CIT shRNA lentivirus and EGFR shRNA lentivirus simultaneously infected human colon cancer RKO cells for 5 days, significantly inhibiting cell proliferation

detailed description

The present invention is further set forth below in conjunction with embodiment. It should be understood that the examples are only used to illustrate the present invention, not to limit the scope of the present invention. The experimental method and the reagent of unspecified formula are all according to conventional conditions in the embodiment, such as [US] Sambrook.J et al.; Huang Peitang etc. translation. Molecular Cloning Experimental Guide, Third Edition. Beijing: The conditions described in Science Press 2002 or the conditions suggested by the manufacturer were performed or configured.

Example 1 Preparation of RNAi lentivirus for human CIT gene and EGFR gene

1. Lentiviral vector construction

1) Construction of lentiviral vectors targeting human CIT gene and EGFR gene

The information of CIT (NM_007174) and EGFR gene (NM_201282) was retrieved from Genbank; effective siRNA targets for CIT gene and EGFR gene were designed using the design software Genechem of Shanghai Jikai Gene Chemical Technology Co., Ltd. The preliminary screening obtained the siRNA target sequence (SEQ ID NO.1) with obvious function of inhibiting CIT gene expression and the siRNA target sequence (SEQ ID NO.2) with obvious function of inhibiting EGFR gene expression. BLAST analysis was carried out through the Genbank database, and it was determined that it did not interfere with any other genes.

Table 1 siRNA target sequence

SEQ ID NO. TargetSeq start site target gene 1 GTCCTCATACCAGGATAAA 5709 CIT 2 GGCTGGTTATGTCCTCATT 499 EGFR

For the siRNA target (SEQ ID NO: 1), synthesize a double-stranded DNA Oligo sequence (Table 2) with Age I and EcoR I restriction sites at both ends; act on GV115 with Age I and EcoR I restriction endonucleases Carrier (carrier with green fluorescent marker, provided by Shanghai Jikai Gene Chemical Technology Co., Ltd., Figure 1), to make it linear.

For the siRNA target (SEQ ID NO: 2), a double-stranded DNA Oligo sequence containing Age I and EcoR I restriction site sticky ends at both ends was synthesized (Table 3). Use Age I and EcoR I restriction enzymes to act on the GV113 vector (carrier with red fluorescent marker, provided by Shanghai Jikai Gene Chemical Technology Co., Ltd., Figure 2) to make it linear.

Table 2 Double-stranded DNA Oligo with sticky ends containing Age I and EcoR I restriction sites at both ends

Table 3 Double-stranded DNA Oligo with sticky ends containing Age I and EcoR I restriction sites at both ends

Ligate the vector DNA and the purified double-stranded DNA Oligo with the double-digested linearized (enzyme digestion system as shown in Table 4, 37°C, reaction for 1 h) by T4 DNA ligase, and in an appropriate buffer system (the ligation system is as shown in Table 5) shown) at 16°C for overnight ligation, and the ligated product was recovered. The ligation product was transformed into fresh Escherichia coli competent cells prepared with calcium chloride (transformation operation reference: Molecular Cloning Experiment Guide, Second Edition, pages 55-56).

Dip it on the surface of the bacterial clone grown by the connection transformation product, dissolve it in 10 μl LB medium, mix well and take 1 μl as a template; in the upstream and downstream of the RNAi sequence in the lentiviral vector, design the upstream primer sequence of the general PCR primer: 5'-CCTATTTCCCATGATTCCTTCATA -3' (SEQ ID NO: 7); downstream primer sequence: 5'-GTAATACGGTTATCCACGCG-3' (SEQ ID NO: 8), for PCR identification experiment (PCR reaction system is shown in Table 6, and reaction conditions are shown in Table 7). The PCR-identified positive clones were sequenced and analyzed, and the PCR identification results are shown in Figure 3 and Figure 4. Correctly aligned clones were successfully constructed RNAi vectors containing SEQ ID NO: 1 or SEQ ID NO: 2, named CIT-shRNA-plasmid and EGFR-shRNA-plasmid, respectively.

2) Construction of GV115 negative control plasmid

The negative control siRNA target sequence was 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 13). When constructing a negative control plasmid, synthesize a double-stranded DNA Oligo sequence containing Age I and EcoR I restriction sites at both ends for the Scr siRNA target (Table 8), and the rest of the construction method, identification method and conditions are the same as CIT-shRNA-plasmid and EGFR-shRNA-plasmid.

Table 4 vector plasmid digestion reaction system

Table 5 Carrier DNA and double-stranded DNA Oligo ligation reaction system

Reagent Positive control (μl) Self-linked control (μl) Connectome (μl) Linearized carrier DNA (100ng/μl) 1.0 1.0 1.0 Annealed dsDNA Oligo (100ng/μl) 1.0 - 1.0 10x T4 phage DNA ligase buffer 1.0 1.0 1.0 T4 bacteriophage DNA ligase 1.0 1.0 1.0 dd H ₂ O 16.0 17.0 16.0 Total 20.0 20.0 20.0

Table 6 PCR reaction system

Reagent Volume (μl) 10×buffer 2.0 dNTPs (2.5mM) 0.8 upstream primer 0.4 downstream primer 0.4 Taq polymerase 0.2 template 1.0 ddH ₂ O 15.2 Total 20.0

Table 7 PCR reaction system program setting

Table 8 Double-stranded DNA Oligo with sticky ends containing Age I and EcoR I restriction sites at both ends

SEQ ID 5' neck ring neck 3' 14 chain of justice CCGG TTCTCCGAACGTGTCACGT CTCGAG ACGTGACACGTTCGGAGAA TTTTTG 15 antisense strand AATTCAAAAA TTCTCCGAACGTGTCACGT CTCGAG ACGTGACACGTTCGGAGAA

2. Packaging to obtain CIT gene interference lentivirus and EGFR gene interference lentivirus

1) CIT gene interference lentivirus

The RNAi plasmid CIT-shRNA-plasmid was extracted with Qiagen's plasmid extraction kit to prepare a 100ng/μl storage solution. 24 hours before transfection, human embryonic kidney cell 293T cells in the logarithmic growth phase were digested with trypsin, and the cell density was adjusted to 1.5×10 ⁵ cells/ml with DMEM complete medium containing 10% fetal bovine serum, and seeded in a 6-well plate , 37°C, 5% CO ₂ incubator. When the cell density reaches 70%-80%, it can be used for transfection. 2 hours before transfection, aspirate the original medium and add 1.5ml fresh complete medium.

According to the instructions of the MISSION Lentiviral Packaging Mix kit from Sigma-aldrich, add 20 μl of Packing Mix (PVM), 12 μl of PEI, and 400 μl of serum-free DMEM medium to a sterilized centrifuge tube, take 20 μl of the plasmid DNA extracted above, and add To the above PVM/PEI/DMEM mixture. The above transfection mixture was incubated at room temperature for 15 min, transferred to the culture medium of human embryonic kidney cell 293T cells, and cultured in a 5% CO ₂ incubator at 37° C. for 16 h. Discard the culture medium containing the transfection mixture, wash with PBS solution, add 2ml of complete medium, and continue to cultivate for 48h.

The cell supernatant was collected, and the Centricon Plus-20 centrifugal ultrafiltration device (Millipore) was used to purify and concentrate the lentivirus. The steps were as follows: (1) 4°C, 4000g centrifugation for 10 min to remove cell debris; (2) Filter the supernatant with a 0.45 μm filter In a 40ml ultracentrifuge tube; (3) 4000g centrifugation, 10-15min, to the required virus concentration volume; (4) After centrifugation, separate the filter cup from the filtrate collection cup below, and buckle the filter cup upside down On the sample collection cup, centrifuge for 2 minutes and the centrifugal force does not exceed 1000g; (5) Remove the centrifuge cup from the sample collection cup, and the CIT gene interference lentivirus concentrate is in the sample collection cup. Store the virus concentrate at -80°C after aliquoting.

2) The packaging process of the EGFR gene interference lentivirus is the same as that of the CIT gene interference lentivirus.

3) The negative control lentivirus packaging process is the same as the CIT gene interference lentivirus.

Example 2 The synergistic effect of CIT gene interference lentivirus and EGFR gene interference lentivirus on tumor treatment

1. Experimental method

Human colon cancer RKO cells in the logarithmic growth phase were digested with trypsin, made into a cell suspension (the number of cells was about 5×10 ⁴ /ml) and inoculated in a 6-well plate, and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI of RKO is 5), the experimental group and the control group were set at the same time. The experimental group used 0.03uL titer of 5×10 ⁸ TU/mL CIT gene interference lentivirus and 0.03uL titer of 5× 10 ⁸ TU/mL EGFR gene interference lentivirus was added to the cancer cell culture medium, and 0.03 uL of the control virus with a titer of 5×10 ⁸ TU/mL was added to the cancer cell culture medium in the control group.

After 24 hours of culture, the medium was replaced, and after the infection time reached 5 days, the cells of each experimental group and control group in the logarithmic growth phase were collected. The complete medium was resuspended into a cell suspension (2×10 ⁴ /ml), and seeded in a 96-well plate at a cell density of about 2000 cells/well. 5 replicate wells in each group, 100 μl per well. After laying the plates, culture them in a 37°C, 5% CO ₂ incubator. From the second day after plating, the Cellomics ArrayScan VTI high-content screening analyzer (Thermo Fisher) was used to detect and read the plate once a day, and the plate was continuously detected and read for 4 days. By adjusting the input parameters of Cellomics arrayscan, accurately calculate the number of cells with green fluorescence and red fluorescence in each scanning plate in each experimental group and control group (simultaneously infected with CIT gene interference lentivirus and EGFR gene interference lentivirus Virus cells), the number of cells with green fluorescence alone (cells infected only with CIT gene interference lentivirus), the number of cells with red fluorescence alone (cells infected with EGFR gene interference lentivirus only), statistical drawing of the data, Draw the cell proliferation curve.

The results of the cell proliferation curve are shown in Figure 5. The results show that: after 4 days in vitro culture of human colon cancer RKO cells infected with CIT shRNA lentivirus and EGFR shRNA lentivirus, the proliferation ratio of viable cells decreased by 68.23% compared with the control group. The RKO cells infected with EGFR shRNA lentivirus were cultured in vitro for 4 days, and the proliferation multiples of viable cells decreased by 11.28% compared with the control group. Compared with the control group, it decreased by 45.94%. , the results of this experiment showed that the cell growth inhibition rate after double gene knockdown was significantly greater than the sum of single gene knockdown groups, indicating that the CIT gene shRNA lentivirus can cooperate with the EGFR gene shRNA lentivirus to inhibit the proliferation of tumor cells.

Claims (6)

1. Use of the isolated human CIT gene and human EGFR gene together as a drug or preparation targeting RNA interference targets of tumor cells in the preparation or screening of tumor therapeutic drugs; As the object of action, the drug is screened to find a drug that can inhibit the expression of the human CIT gene and a drug that inhibits the expression of the human EGFR gene, and then use it as a candidate composition drug for tumor treatment; the tumor is colon cancer; the CIT gene The target sequence of the EGFR gene is the sequence shown in SEQ ID NO:1; the target sequence of the EGFR gene is the sequence shown in SEQ ID NO:2. 2. A nucleic acid molecule drug for treating colon cancer, its active ingredient contains an isolated nucleic acid molecule that reduces the expression of CIT gene in tumor cells, and an isolated nucleic acid molecule that reduces the expression of EGFR gene in tumor cells; The isolated nucleic acid molecule that reduces CIT gene expression in tumor cells comprises: a) CIT gene double-stranded RNA, which contains a nucleotide sequence capable of hybridizing with the CIT gene under stringent conditions; or b) CIT gene shRNA, which contains a nucleotide sequence capable of hybridizing with the CIT gene under stringent conditions; The isolated nucleic acid molecule that reduces EGFR gene expression in tumor cells comprises: A. EGFR gene double-stranded RNA, which contains a nucleotide sequence capable of hybridizing with the EGFR gene under stringent conditions; or B. EGFR gene shRNA, which contains a nucleotide sequence capable of hybridizing with the EGFR gene under stringent conditions; The CIT gene double-stranded RNA comprises a first strand and a second strand, and the first strand and the second strand are complementary to form an RNA dimer; the CIT gene shRNA includes a sense strand fragment and an antisense strand fragment, and a stem-loop structure connecting the sense strand fragment and the antisense strand fragment, the sequences of the sense strand fragment and the antisense strand fragment are complementary; the EGFR gene double-stranded RNA comprises a first strand and a second strand, so The first strand and the second strand are complementary to form an RNA dimer together; the EGFR gene shRNA includes a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, The sequences of the sense strand fragment and the antisense strand fragment are complementary; the target sequence of the CIT gene is the sequence shown in SEQ ID NO:1; the target sequence of the EGFR gene is the sequence shown in SEQ ID NO:2 . 3. The nucleic acid molecule drug according to claim 2, wherein the double-stranded RNA is a small interfering RNA, and the sequence of the first strand of the CIT gene small interfering RNA is as shown in SEQ ID NO: 9; the EGFR gene small interfering RNA The sequence of the first strand of the interfering RNA is shown in SEQ ID NO: 10; the sequence of the CIT gene shRNA is shown in SEQ ID NO: 11; the sequence of the EGFR gene shRNA is shown in SEQ ID NO: 12. 4. A lentiviral vector drug for treating tumors, the active ingredient of which contains a CIT gene interference lentiviral vector and an EGFR gene interference lentiviral vector, and the CIT gene interference lentiviral vector contains the encoding content of any one of claims 2-3. A gene segment of the CIT gene shRNA in the nucleic acid molecule drug; the EGFR gene interference lentiviral vector contains a gene segment encoding the EGFR gene shRNA in the nucleic acid molecule drug of any one of claims 2-3. 5. A lentiviral drug for treating tumors, its active ingredient contains CIT gene interference lentivirus and EGFR gene interference lentivirus, the CIT gene interference lentivirus is composed of the CIT gene interference lentivirus in the lentiviral carrier drug described in claim 4 The viral vector is formed through viral packaging with the assistance of lentiviral packaging plasmids and cell lines; the EGFR gene interference lentivirus consists of the EGFR gene interference lentiviral vector in the lentiviral vector medicine described in claim 4 in the lentiviral packaging plasmid , with the help of cell lines, it is packaged by virus. 6. A pharmaceutical composition for the treatment of colon cancer, its composition contains the isolated nucleic acid molecules that reduce the expression of CIT gene in tumor cells in the nucleic acid molecule medicine according to any one of claims 2-3, the slow-growing enzymes described in claim 4 At least one of the CIT gene interference lentivirus vector in the viral vector drug, the CIT gene interference lentivirus in the lentiviral drug described in claim 5; and the nucleic acid molecule drug described in any one of claims 2-3 At least one of the isolated nucleic acid molecule that reduces EGFR gene expression in tumor cells, the EGFR gene interference lentiviral vector in the lentiviral vector medicine described in claim 4, and the EGFR gene interference lentivirus in the lentiviral medicine described in claim 5 kind.