Application of combination of CCT6A gene and p53 gene in preparation of intestinal cancer gene therapy drug

Abstract

The invention discloses application of combination of a CCT6A gene and a p53 gene in preparation of a gene therapy drug for intestinal cancer, and belongs to the field of gene therapy, the gene therapy drug simultaneously regulates and controls the CCT6A gene and a wild type p53 gene, and the intestinal cancer is that the CCT6A gene is abnormally high in expression and the p53 gene is mutated. The invention provides a treatment thought aiming at CCT6A high expression and p53 mutation carrying tumors, and the accuracy of tumor treatment can be improved.

Description

Technical Field

[0001] The present invention is an application of CCT6A gene and p53 gene in the preparation of colorectal cancer gene therapy drugs, specifically relates to a drug for the treatment of colorectal cancer with high expression of CCT6A gene and p53 gene mutation using a combination of CCT6A interference and wild-type p53 overexpression genes, belonging to the field of gene therapy. Background Art

[0002] Cancer is one of the most serious diseases that threaten human life and health. Traditional cancer treatments usually include general treatments such as surgery, chemotherapy, and radiotherapy. In recent years, with the continuous development of biotechnology, whole genome sequencing is used to analyze the heterogeneity of tumor patients, and personalized drugs are designed for abnormal genes related to cancer to achieve precision treatment. Compared with traditional treatments, it can effectively improve the treatment effect and patient survival rate.

[0003] In the human genome, p53 is a very important tumor suppressor gene and the gene with the highest mutation frequency in human tumors. More than 50% of cancer patients have p53 gene mutations. Wild-type p53 is an important regulator of cell cycle, DNA damage repair, and cell apoptosis, which can prevent abnormal cell proliferation. However, in addition to losing the tumor suppressor function of wild-type p53, mutant p53 can also gain other functions to promote tumor cell proliferation, survival, migration, and drug resistance. The development of p53-based anti-tumor drugs is divided into two aspects: one is to restore the activity of wild-type p53 in cancer cells; the other is to eliminate the acquired functional activity of mutant p53 in cancer cells. TRiC (TCP1 ring complex) participates in protein folding in cells and regulates the expression of multiple tumor-related proteins and the cell cycle. CCT6A is a member of the TRiC family. There is no report on the combined treatment of tumors by simultaneously regulating CCT6A and p53 genes. Summary of the invention

[0004] The purpose of the present invention is to provide an application of CCT6A gene and p53 gene in the preparation of colorectal cancer gene therapy drugs. When used in tumor gene therapy, the accuracy of tumor treatment can be improved.

[0005] The present invention is achieved through the following technical scheme: the combined use of CCT6A gene and p53 gene in the preparation of a gene therapy drug for colorectal cancer, wherein the gene therapy drug simultaneously regulates the CCT6A gene and the wild-type p53 gene, and the colorectal cancer is characterized by abnormally high expression of the CCT6A gene and mutation of the p53 gene.

[0006] The regulation is to use a lentiviral vector, an adenoviral vector or an adeno-associated viral vector to down-regulate the CCT6A gene and up-regulate the wild-type p53 gene.

[0007] The lentiviral vector is constructed by constructing the interference sequence of the CCT6A gene and the sequence of the wild-type p53 gene into a lentiviral expression vector, and has the dual gene regulation function of simultaneously down-regulating the CCT6A gene and up-regulating the wild-type p53 gene.

[0008] The combination of CCT6A gene and p53 gene refers to the combined expression of the interference sequence of CCT6A gene and the wild-type p53 gene.

[0009] For example, a dual gene recombinant vector of CCT6A interference (shCCT6A) and wild-type p53 (Wtp53) overexpression is provided, wherein the recombinant vector is a vector that simultaneously expresses shCCT6A and Wtp53.

[0010] The vector includes a lentiviral vector, an adenoviral vector or an adeno-associated viral vector, thereby obtaining a shCCT6A+Wtp53 double gene recombinant lentiviral vector, a shCCT6A+Wtp53 double gene recombinant adenoviral vector or a shCCT6A+Wtp53 double gene recombinant adeno-associated viral vector. The construction method of the lentiviral recombinant vector is as follows: constructing the shCCT6A sequence and the Wtp53 (wild-type p53) sequence into the lentiviral expression vector Lv-sh-oe-ZsGreen-puro to obtain the recombinant lentiviral vector Lv-shCCT6A- Wtp53-ZsGreen-puro.

[0011] The recombinant lentiviral vector has dual gene regulatory functions of down-regulating CCT6A and up-regulating wild-type p53.

[0012] The above-mentioned dual gene combination of CCT6A interference and wild-type p53 overexpression can lay the foundation for the development of gene therapy drugs in the treatment of colorectal cancer with high expression of CCT6A gene and carrying p53 gene mutation.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The present invention is the first to significantly inhibit the proliferation of colorectal cancer cells by downregulating CCT6A and upregulating wild-type p53. The inhibitory effect on colorectal cancer is greater than that of downregulating CCT6A or upregulating wild-type p53 alone.

[0014] (2) The present invention provides a treatment strategy for colorectal cancer with high expression of CCT6A and p53 mutation. By providing a drug with dual gene regulatory functions of downregulating CCT6A and upregulating wild-type p53, the precision of tumor treatment can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1This is a multivariate survival analysis of CCT6A expression and p53 status in colorectal cancer samples.

[0016] Figure 2 Schematic diagram of the recombinant lentiviral vector structure for simultaneously downregulating the CCT6A gene and upregulating the wild-type p53 gene.

[0017] Figure 3 The combined expression of shCCT6A+Wtp53 can significantly inhibit tumor cell growth in vitro.

[0018] Figure 4 The combined expression of shCCT6A+Wtp53 can significantly inhibit tumor growth in vivo. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0020] Example 1: Evaluation of the effect of high CCT6A expression and p53 mutation on the overall survival of colorectal cancer patients 1. Data processing Clinical information of 594 colorectal cancer patients was obtained from the TCGA database. The samples were divided into CCT6A low expression + p53 wild group, CCT6A high expression + p53 wild group, CCT6A low expression + p53 mutation group and CCT6A high expression + p53 mutation group. Survival analysis was performed based on the survival time and survival events of the patients. The four groups of patients were compared using Kaplan-Meier survival plots, and P values ​​were calculated.

[0021] 2. Results Analysis like Figure 1 As shown, the overall survival of colorectal cancer patients in the CCT6A low expression + p53 wild group was higher than that in the CCT6A high expression + p53 mutation group, and P value <0.05 indicated statistical significance.

[0022] Example 2: Construction of shCCT6A+Wtp53 recombinant lentiviral vector and preparation of lentivirus (see Figure 2 Structure shown) (I) Construction of shCCT6A sequence into Lv-sh-oe-ZsGreen1-puro vector (1) Vector digestion The enzyme digestion system is: Lv-sh-oe-ZsGreen1-puro plasmid, 10ul; endonuclease reaction buffer, 2ul; restriction endonuclease, 1ul; ddH2O, 7ul. After mixing, place at 37℃ for 1-2h. Proceed to the next step immediately or store at -20℃.

[0023] (2) Electrophoresis detection The digestion products were subjected to agarose gel electrophoresis to check whether the digestion was successful. Non-target fragments were removed by gel extraction and then purified using the OMEGA gel extraction kit.

[0024] (3) Preparation of shCCT6A DNA fragment The annealing primers for the shCCT6A DNA fragment are: shCCT6AF1: 5'-GATCGAAGGGCACCATGAAGATGCTCCTCGAGGAGCATCTTCATGGTGCCCTTTTTTTG-3', as shown in SEQ ID NO.1; shCCT6AR2: 5'-AATTCAAAAAAAGGGCACCATGAAGATGCTCCTCGAGGAGCATCTTCATGGTGCCCTTC-3', as shown in SEQ ID NO.2.

[0025] The annealing reaction system is: shCCT6AF1 (10μM), 2μl; shCCT6AR2 (10μM), 2μl; ddH2O, 16μl. After thorough mixing, put it into the thermal cycler, and the reaction program is: 37℃, 30min; 95℃, 5min, and then cool to 25℃ at 5℃ / min. After annealing, it is used for ligation reaction.

[0026] (4) Connecting the vector to the target gene The ligation system is: vector, 0.5ul; shCCT6A DNA fragment, 1.5ul; ligase, 0.5ul; ligation buffer, 1ul; ddH2O, 6.5ul. After ligation at 24℃ for 1h, add 100 μl of E. coli competent cells. Ice bath for 30min, heat shock at 42℃ for 90s, ice bath for 2min, do not shake during this period. Add 800μl of 37℃ preheated SOC medium (without antibiotics), culture at 37℃ for 1h, then spread on LB agar plate containing Amp, and culture at 37℃ for 12-16h.

[0027] (5) Identification of positive clones Add 800μl of LB liquid culture medium containing Amp to a 1.5ml centrifuge tube, pick a single clone colony in the centrifuge tube, and culture at 37℃ for 6-7h before performing colony PCR identification. The identification system is: interference plasmid detection taq enzyme Mix, 6ul; bacterial liquid, 2.5ul, ddH2O 3.5μl. After thorough mixing, centrifuge for 1-2s and then perform PCR reaction. The reaction procedure is shown in Table 1 below.

[0028] Table 1 , The PCR products were analyzed by agarose gel electrophoresis. A band of about 219 bp was detected in the positive clones, and about 150 bp in the negative clones or negative control groups. The positive clones were sequenced for verification, and the sequencing primers were: the universal primers for human U6 promoter prepared by the sequencing company.

[0029] (II) Construction of Wtp53 sequence into Lv-shCCT6A-oe-ZsGreen-puro vector According to the results of the monoclonal detection, the bacteria containing the positive plasmid continued to be cultured. The bacteria contained the Lv-shCCT6A-oe-ZsGreen-puro plasmid. The plasmid of the corresponding bacterial solution was extracted using the OMEGA plasmid extraction kit. The Wtp53 sequence was constructed based on this plasmid. The steps are as follows: (1) Vector digestion The enzyme digestion system is: Lv-shCCT6A-oe-ZsGreen-puro plasmid, 10ul; endonuclease reaction buffer, 2ul; restriction endonuclease, 1ul; ddH2O, 7ul. After fully mixing, place at 37℃ for 1-2h. Proceed to the next step immediately or store at -20℃.

[0030] (2) Electrophoresis detection The digestion product was subjected to agarose gel electrophoresis to check whether the digestion was successful. Non-target fragments were removed by gel extraction and then purified using the OMEGA gel extraction kit. (3) Preparation of Wtp53 DNA fragment The amplification primers for the Wtp53 DNA fragment were: Wtp53F1: 5'-AGCAGCGGCCGCCACCATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCCTC-3', as shown in SEQ ID NO.3; Wtp53R2: 5'-GCCCCCGGGGTCTGAGTCAGGCCCTTCTGTCTTGAACATGAGTTTT-3', as shown in SEQ ID NO.4.

[0031] The amplification system of Wtp53 DNA fragment is: cDNA template, 1ul; Wtp53F1 (10μM), 2μl; Wtp53R2 (10μM), 2μl; PCRmix, 15ul; ddH2O, 6.5ul. The reaction procedure is the same as Table 1.

[0032] The PCR product was analyzed by agarose gel electrophoresis, and a band of about 1200 bp was detected. The non-target fragments were removed by gel extraction and then purified using the OMEGA gel extraction kit. The purified fragments were digested with enzymes, and the digestion system was: Wtp53 DNA fragment, 10ul; endonuclease reaction buffer, 2ul; restriction endonuclease, 1ul; ddH2O, 7ul. After thorough mixing, place at 37℃ for 1 to 2h. Proceed to the next step immediately or store at -20℃.

[0033] The digested product was subjected to agarose gel electrophoresis to check whether the digestion was successful. Non-target fragments were removed by gel extraction and then purified using the OMEGA gel extraction kit.

[0034] (3) Connecting the vector to the target gene The ligation system is: vector, 0.5ul; Wtp53 DNA fragment after enzyme digestion, 1.5ul; ligase, 0.5ul; ligation buffer, 1ul; ddH2O, 6.5ul. After ligation at 24℃ for 1h, add 100 μl of E. coli competent cells. Ice bath for 30min, heat shock at 42℃ for 90s, ice bath for 2min, do not shake during this period. Add 800μl of SOC medium (without antibiotics) preheated at 37℃, culture at 37℃ for 1h, then spread on LB agar plate containing Amp, and culture at 37℃ for 12-16h.

[0035] (4) Identification of positive clones Add 800μl of LB liquid culture medium containing Amp to a 1.5ml centrifuge tube, pick a single clone colony in the centrifuge tube, and culture at 37℃ for 6-7h before performing colony PCR identification. The identification system is: Taq enzyme Mix (2X), 5ul; p53 primer, 1ul; bacterial solution, 2.5ul; ddH2O, 1.5μl. After thorough mixing, centrifuge for 1-2s before performing PCR reaction. The reaction system is shown in Table 1.

[0036] The PCR products were analyzed by 1% agarose gel electrophoresis. A band of about 1180 bp was detected in the positive clones, while no band was detected in the negative clones or negative control group. The positive clones were sequenced for verification, and the sequencing primers were: universal primers for human EF1a promoter prepared by the sequencing company.

[0037] 3. Lentiviral packaging (1) Preparation of 293T cells The 293T cells with good growth status were subcultured so that the cell density could reach about 70% after 24 hours of culture and could be used for transfection.

[0038] (2) Plasmid transfection The plasmids required for the three-plasmid system for lentiviral packaging are: interfering lentiviral vector, auxiliary plasmid 1 and auxiliary plasmid 2. These three plasmids were sequentially transferred into 293T cells at a concentration ratio of 1:0.75:0.25 using a transfection reagent, and the specific transfection steps were carried out according to the instructions of the transfection reagent used.

[0039] (3) Lentivirus Harvest 48 hours after transfection, collect the cell culture fluid, add 1 ml of virus concentrate to every 4 ml of culture fluid, mix and store in a 4°C refrigerator, mix every 20 minutes, and repeat 3-5 times. Add fresh culture fluid to 293T cells, continue to culture for 24 hours, collect the culture fluid according to the above method, and store at 4°C overnight.

[0040] (4) Lentivirus concentration and purification The mixture of virus / lentivirus concentrate collected twice was centrifuged at 4°C, 7000g for 30 minutes, and a precipitate was visible. The supernatant was discarded, the residual liquid was aspirated, and an appropriate amount of lentivirus preservation solution was added. The precipitate was dissolved by repeated blowing and beating. After the virus solution was filtered with a pore size of 0.45µm, the virus was further purified and concentrated by sucrose gradient centrifugation and ultrafiltration, and stored in aliquots at -80°C.

[0041] Example 3: Cell clone formation experiment verifies the effect of shCCT6A combined with Wtp53 on colorectal cancer cells 1. Crystal violet staining Fix the cells with anhydrous ethanol for 10 to 20 minutes, then rinse twice with distilled water for 2 to 5 minutes each time. Add an appropriate volume of crystal violet staining solution, incubate at room temperature for 10 to 30 minutes, wash twice with distilled water for 2 minutes each time, and perform staining analysis with Image J software.

[0042] 2. Results Analysis like Figure 3 As shown in the figure, compared with the control group, the shCCT6A+Wtp53 group significantly inhibited cell proliferation, and the effect was greater than that of the control group, shCCT6A group and Wtp53 group. The difference was statistically significant (P<0.05).

[0043] Example 4: Nude mouse tumor formation experiment verifies the effect of shCCT6A combined with Wtp53 on xenograft tumors 1. Nude mouse xenograft tumor model Twelve 4-6 week old nude mice were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. and divided into control group, shCCT6A group, Wtp53 group, and shCCT6A+Wtp53 group. Each group was subcutaneously inoculated with 1×10 7The corresponding lentivirus was used to infect stable SW480 cells (SW480 cells are colorectal cancer cells with high expression of CCT6A and p53 mutation), and the growth status of nude mice was observed every other day.

[0044] 2. Tumor volume monitoring The longest diameter (a) and shortest diameter (b) of the tumor were measured with a vernier caliper every 5 days. The tumor volume was calculated using the formula V=ab 2 / 2. The experiment ended on day 25 and the tumor weight was measured. This experiment followed the ethical review guidelines for experimental animal welfare.

[0045] (III) Experimental results like Figure 4 As shown, the growth rate and tumor volume of the shCCT6A+Wtp53 group were lower than those of the control group, shCCT6A group, and Wtp53 group. The experiment showed that the shCCT6A+Wtp53 recombinant lentivirus can effectively inhibit the growth of xenograft tumors in nude mice. This shows that the technical solution of the present invention can be applied to the treatment of colorectal cancer with high expression of CCT6A and p53 mutation.

[0046] In summary, in Example 1, the present invention first discovered that the overall survival of colorectal cancer patients in the CCT6A low expression + p53 wild group was higher than that in the CCT6A high expression + p53 mutation group. In Example 2, the present invention first combined shCCT6A and wild-type p53 to construct a shCCT6A + Wtp53 double gene recombinant vector, which can simultaneously achieve CCT6A gene downregulation and wild-type p53 gene upregulation, targeting colorectal cancer with high CCT6A expression and p53 mutation. In Examples 3 and 4, it was verified at the cell level and animal experimental level that the combined treatment of downregulating CCT6A and upregulating Wtp53 can effectively inhibit the tumor growth of colorectal cancer cells and colorectal cancer xenograft animal models.

[0047] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. Application of CCT6A gene and p53 gene in the preparation of gene therapy drugs for colorectal cancer. Features: The gene therapy drug is used to simultaneously regulate the CCT6A gene and the wild-type p53 gene. The intestinal cancer is caused by abnormally high expression of the CCT6A gene and mutation of the p53 gene.

2. The use according to claim 1, Features: The regulation is to use a lentiviral vector, an adenoviral vector or an adeno-associated viral vector to down-regulate the CCT6A gene and up-regulate the wild-type p53 gene.

3. The use according to claim 2, Features: The lentiviral vector is constructed by constructing the interference sequence of the CCT6A gene and the sequence of the wild-type p53 gene into a lentiviral expression vector, and has the dual gene regulation function of simultaneously down-regulating the CCT6A gene and up-regulating the wild-type p53 gene.

4. The use according to claim 1, Features: The combination of CCT6A gene and p53 gene refers to the combined expression of the interference sequence of CCT6A gene and the wild-type p53 gene.

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