MSANTD2 gene and its RNAi lentiviral interference system and application

Abstract

The present invention discloses the MSANTD2 gene and its RNAi lentiviral interference system and application, and relates to the field of biomedical technology. The application of the MSANTD2 gene disclosed in the present invention in the preparation of a drug for treating colorectal cancer, wherein the treatment of colorectal cancer is to inhibit the proliferation activity and migration and invasion ability of colorectal cancer cells. The MSANTD2 gene is used as a target to inhibit the expression of MSANTD2 protein in colorectal cancer cells, an RNAi lentiviral interference system of the MSANTD2 gene is constructed, and a drug for treating colorectal cancer is prepared, and a kit for diagnosing diseases with high expression of the MSANTD2 gene / protein in colorectal cancer is prepared. It provides a new strategy and direction for the treatment of colorectal cancer, has a very high clinical application value, and is expected to bring better therapeutic effects to patients with intestinal cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to a MSANTD2 gene and an RNAi lentiviral interference system and application thereof. Background Art

[0002] Colorectal cancer (CRC) is the third most common malignant tumor in the world and the second leading cause of cancer-related death. Although the combined application of multiple treatment methods such as surgical resection, radiotherapy / chemotherapy, targeted therapy and immunotherapy has improved the overall survival rate of CRC patients, the clinical prognosis is still poor due to the high incidence of metastasis and recurrence in patients with colorectal cancer diagnosed in the late stage. The formation and development of colorectal cancer is a complex process involving changes in many molecules and signaling pathways, and its mechanism has not yet been fully elucidated. Therefore, in-depth research on the molecular mechanism of the occurrence and development of colorectal cancer and the search for feasible molecular diagnostic and therapeutic targets are of great significance to improve the survival rate of patients.

[0003] The MSANTD2 gene is located on chromosome 11 of the human genome, with a total length of 2384 bases (bp), and 4 exons encoding a protein of 559 amino acids (aa). Currently, no research on MSANTD2 and malignant tumors has been reported. Summary of the invention

[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide an RNAi lentiviral interference system of the MSANTD2 gene. Another technical problem to be solved by the present invention is to provide an application of the RNAi lentiviral interference system of the MSANTD2 gene for preparing a drug for treating colorectal cancer.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] Use of the MSANTD2 gene in preparing a drug for treating colorectal cancer.

[0007] The method for treating colorectal cancer is to inhibit the proliferation activity of colorectal cancer cells.

[0008] The method for treating colorectal cancer is to inhibit the migration ability of colorectal cancer cells.

[0009] The method for treating colorectal cancer is to inhibit the invasive ability of colorectal cancer cells.

[0010] The biomarker used for diagnosis, treatment or prognosis of colorectal cancer is the MSANTD2 gene.

[0011] A kit for diagnosis, treatment or prognosis of colorectal cancer, comprising a MSANTD2 gene sequence.

[0012] Use of the RNAi lentiviral interference system of the MSANTD2 gene in the preparation of a drug for treating colorectal cancer, the sequence of the RNAi lentiviral interference system of the MSANTD2 gene is as follows:

[0013] MSANTD2-shRNA1: 5'-TTTGAGCAAGCAAGATATATA-3',

[0014] MSANTD2-shRNA2: 5'-ACTCTACAGCAGGTGCTTATTT-3'.

[0015] The RNAi lentiviral interference system of the MSANTD2 gene uses the MSANTD2 gene as a target to inhibit the expression of MSANTD2 protein in colorectal cancer cells.

[0016] The RNAi lentiviral interference system of the MSANTD2 gene uses the MSANTD2 gene as a target to knock out the MSANTD2 gene in colorectal cancer cells.

[0017] The RNAi lentiviral interference system of the MSANTD2 gene uses the MSANTD2 gene as a target to silence the MSANTD2 gene in colorectal cancer cells.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1) The MSANTD2 gene / protein disclosed in the present invention is a target for precision treatment of colorectal cancer, and a drug for treating colorectal cancer and a kit for diagnosing diseases with high expression of the MSANTD2 gene / protein in colorectal cancer are prepared to inhibit the proliferation activity, migration ability and invasion ability of colorectal cancer cells. It provides a new strategy and direction for the treatment of colorectal cancer, has extremely high clinical application value, and is expected to bring better treatment effects to patients with colorectal cancer.

[0020] 2) The expression level of the MSANTD2 gene disclosed in the present invention is significantly increased in colorectal cancer tissues and cells, and thus can be used as an effective biomarker for the diagnosis of colorectal cancer.

[0021] 3) The RNAi lentiviral interference system of the MSANTD2 gene disclosed in the present invention efficiently knocks down the MSANTD2 gene / protein in colorectal cancer cells, inhibits the proliferation, invasion and migration of cancer cells, and the system is simple to operate and highly efficient.

[0022] 4) The relative protein expression of MSANTD2 in DLD1 cells and SW1116 cells treated with the RNAi lentiviral interference system of the MSANTD2 gene disclosed in the present invention was significantly reduced compared with that in the control group, indicating that the expression of MSANTD2 protein in DLD1 cells and SW1116 cells was effectively inhibited.

[0023] 5) After knocking down MSANTD2 using the RNAi lentiviral interference system of the MSANTD2 gene disclosed in the present invention, the proliferation activity, invasion and migration abilities of DLD1 cells and SW1116 cells decreased. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Figure 2 is a graph showing the mRNA expression level of MSANTD2 in colorectal cancer (A is a graph showing the mRNA expression level of MSANTD2 in the TCGA database; B is a graph showing the ROC curve analysis of MSANTD2 expression in colorectal cancer patients);

[0025] Figure 2 The protein expression level of MSANTD2 in colorectal cancer (scale = 50um) (A is the fluorescence staining of MSANTD2 in colorectal cancer tissue; B is the expression of MSANTD2 in colorectal cancer tissue and adjacent tissue detected by Western Blot);

[0026] Figure 3 Western Blot detection of MSANTD2 in colorectal cancer cells (A is the expression diagram of NCM460 cells, DLD1 cells, SW480 cells, CACO2 cells, and SW1116 cells; B is the expression diagram of MSANTD2 in colorectal cancer cells detected by fluorescence quantitative PCR);

[0027] Figure 4 is a diagram of the structure of a lentiviral vector;

[0028] Figure 5 The expression diagram of MSANTD2 after knocking out the MSANTD2 gene (A is the Western Blot detection diagram; B is the fluorescence quantitative PCR detection diagram);

[0029] Figure 6 This is a diagram showing the effect of knocking out the MSANTD2 gene on the proliferation function of colorectal cancer cells;

[0030] Figure 7 This figure shows the effect of knocking out the MSANTD2 gene on the invasion and migration function of colorectal cancer cells. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0032] The main reagents used in the following examples are as follows:

[0033] Opal 7-color immunohistochemistry kit (Perkin Elmer, USA); anti-human MSANTD2 antibody (472864-HRP, Biorbyt, UK) was used for multiple immunofluorescence and immunoblotting experiments; anti-CK antibody was used; colorectal cancer cell lines (DLD1, SW480, CACO2, SW1116) and intestinal epithelial cells (NCM460) were purchased from Nanjing Kebai Biotechnology Co., Ltd. RPMI-1640 medium and fetal bovine serum (Gibco, USA).

[0034] The main instruments used in the following examples are as follows:

[0035] Multispectral pathology scanning system: Perkin Elmer, USA;

[0036] Inverted fluorescence microscope: Zeiss, Germany;

[0037] Gel imaging system: China Tianneng Company;

[0038] Multifunctional microplate reader: Thermo Corporation, USA.

[0039] The tissue chips of colorectal cancer patients used in the following examples are paraffin chips of colorectal cancer stored in the biological sample bank of the Affiliated Hospital of Nantong University (operations were performed between 2012 and 2018, the patients did not receive immunotherapy, chemotherapy or radiotherapy before the operation, and the clinical case data were detailed and complete). There were 79 cases of colorectal cancer tissue and 30 cases of benign tissue.

[0040] Example 1

[0041] 1. The process of making tissue microarray

[0042] 1) Pathological tissue sections

[0043] Fresh tissue blocks (thickness 0.5 cm) removed during surgery were fixed in a pre-prepared 10% formalin solution, and then dehydrated with gradient concentrations of alcohol until xylene was transparent; the transparent tissue blocks were placed in melted paraffin, and embedded after the paraffin was completely immersed in the tissue blocks; after cooling and solidification, they were cut into sections (thickness 5-8 um) and then dried in a 45°C constant temperature box.

[0044] 2) HE staining

[0045] After being immersed in distilled water, the slices were placed in a hematoxylin aqueous solution for staining for several minutes; hydrochloric acid alcohol and ammonia solution were used for colorimetry; after being rinsed with running water for 1 hour, they were placed in distilled water for a while; dehydrated in 70%, 75%, and 90% alcohol for 5 minutes each; they were placed in eosin staining solution for 2-3 minutes; the stained slices were dehydrated with gradient alcohol, and then made transparent with xylene, and neutral gum was dropped on them. After sealing with a coverslip, they were observed under a microscope to determine the tumor area.

[0046] 3) Paraffin and beeswax were mixed in a ratio of 1:1 to prepare blank acceptor wax blocks, and 10×7 holes with a total of 350 tissue arrays were designed on the wax blocks. Then, a TMA blank wax block was prepared using a tissue microarray instrument. According to the microscopic examination results of the HE-stained sections, the most representative tumor area was selected at the marked points on the donor wax block, and a tissue block with a diameter of 2 mm was taken, with one core taken from each case. The taken tissue core was transferred to the hole of the acceptor wax block, and the corresponding non-tumor tissue was taken as a control. The tissue array block was heated and fused in a constant temperature oven at 55°C for 10 minutes, and cooled to room temperature before it was almost melted, so that the acceptor wax block and the donor tissue were integrated. ; Freeze the tissue chip at 4℃ for about 4 hours, then use a fully automatic tissue slicer to trim the tissue array block at a speed of 20mm / rev until all tissue cores are completely exposed; slice the tissue array block with a slicer, float the continuous slices in cold water respectively, let them unfold naturally, then transfer the slices to 45℃ warm water to unfold the slices for about 2 minutes, and after unfolding, stick them on a slide treated with anti-stripping to dry; bake the slices at 60℃ for 3 minutes, and continue to bake at 58℃ for 16h; store the prepared tissue chip in a slice box and store it in a refrigerator at -20℃ for later use.

[0047] 2. Multicolor immunofluorescence histochemistry

[0048] After the tissue chip is placed on a slice dryer, it is dewaxed in xylene, and then dehydrated with gradient alcohol and rinsed with distilled water; then placed in AR6 repair solution with a pH of 6.0, and high-temperature antigen repair is performed on a high-temperature slice rack; after cooling naturally to room temperature, it is rinsed with PBS and blocked with a primary antibody blocking solution for 10 minutes; after completion, 200 μL of anti-human MSANTD2 antibody working solution (dilution ratio of 1:100, biorbyt, UK) is added to the tissue chip and kept at 4°C overnight; the tissue chip is taken out and reheated for 0.5 hours and then rinsed with PBS; then 200 μL of secondary antibody working solution is added to the tissue chip and rinsed with PBS at room temperature for 10 minutes; continue to add the prepared fluorescent dye to the tissue chip, incubate at room temperature for 10 minutes away from light, and then rinse with PBS; after drying until transparent, cover the slice with DAPI. If a second antibody is to be incubated next, high-temperature antigen repair is performed, as described above.

[0049] The staining results were observed under a fluorescence microscope, and staining in the corresponding part of the cell was considered positive. Each sample was captured at 20 times magnification using Vectra3 automatic imaging software. Images were analyzed and scored using inForm4.1.0 (Perkin Elmer), and a threshold of positive or negative cells was set for each cell. The percentage of cells in each area was calculated and scored (0-100), and the cutoff point of MSANTD2 protein expression score was obtained by X-tile software based on survival time and survival status. The scores were as follows: 0-61.49 for low expression or no expression, and 61.5-100 for high expression. All data were processed using SPSS V.22.0 statistical software, and the chi-square test was used for inter-group comparisons. Cox proportional hazard regression was used to analyze the prognostic factors of patients. The Kaplan-Meier method and log-rank test were used for univariate analysis of patient prognosis. All test results were considered statistically significant when P<0.05.

[0050] The results are as follows Figure 1 As shown in Figure 2, compared with normal tissues, the expression level of MSANTD2 mRNA in colorectal cancer was significantly increased ( Figure 1 A); The area under the expression curve (AUC) of MSANTD2 in colorectal cancer was 0.752, and the 95% CI was 0.701-0.804 ( Figure 1 B).

[0051] The results are as follows Figure 2 As shown, multiple immunofluorescence histochemistry and WB also confirmed that the expression level of MSANTD2 in colorectal cancer samples was significantly increased compared with adjacent adjacent cancer tissues, indicating that MSANTD2 can serve as a potential biomarker for the diagnosis of colorectal cancer.

[0052] Example 2

[0053] 1. Culture of colorectal cancer cell lines

[0054] DLD1 cells, SW480 cells, CACO2 cells, and SW1116 cells were cultured in RPMI-1640 complete medium in an incubator maintained at 37°C and 5% CO 2 Routine subculture was performed at saturated humidity, and cells in the logarithmic growth phase were selected for subsequent experiments.

[0055] 2. Extraction of total cell protein

[0056] Collect colon cancer cells in the logarithmic growth phase, discard the culture medium, and wash the cells twice with pre-cooled PBS; add different 1×SDS cell lysis buffers according to the size of the cell culture flask and the growth density of the cells, then scrape the cells with a cell scraper and transfer them to a clean EP tube; fully lyse the scraped cell protein on ice for 30 minutes; centrifuge at 4℃ and keep the supernatant, measure the concentration of cell protein using the BCA method and a multi-function microplate reader, and store in a -20℃ refrigerator for later use.

[0057] 3. Western blot

[0058] Prepare polyacrylamide gel (5% concentrated gel, 10% separation gel); after loading the protein marker and the extracted protein sample, adjust the operating voltage to 100V, and after the end, take out the gel for transfer (PVDF membrane); transfer at a constant current of 300mA for 1.5h, and the transfer must be carried out in an ice box; after the transfer, put the PVDF membrane into the blocking solution and block it at room temperature for 2h; prepare the primary antibody dilution solution with the blocking solution, evenly add the diluted primary antibody to the PVDF membrane, and incubate it at 4℃ overnight; after washing the membrane, prepare the secondary antibody dilution solution with TBST, evenly add the diluted secondary antibody to the PVDF membrane, and incubate it at room temperature for 1.5h; after washing the membrane, spread the PVDF membrane flat on the corresponding position of the developer, dilute the ECL luminescent solution with TBST, evenly add it to the PVDF membrane, and take pictures and save them with the gel imaging system.

[0059] The results are as follows Figure 3 As shown, MSANTD2 was expressed relatively high in DLD1 and SW1116 colorectal cancer cells, and relatively low in CACO2 and SW480 colorectal cancer cells.

[0060] Example 3

[0061] 1. Construction of RNAi lentiviral interference system of MSANTD2 gene

[0062] Aiming at the sequence of MSANTD2 gene (gene ID: 79684, protein ID: Q6P1R3), an RNAi lentiviral interference system specifically targeting the MSANTD2 gene was constructed to construct a lentiviral-mediated RNAi interference system. The gene sequence of the RNAi interference system of the MSANTD2 gene is as follows:

[0063] MSANTD2-shRNA1: 5'-TTTGAGCAAGCAAGATATATA-3',

[0064] MSANTD2-shRNA2: 5'-ACTCTACAGCAGGTGCTTATTT-3'.

[0065] Add a stem-loop structure (CTCGAG) to the above sequence and supplement the palindromic sequence and restriction site. After annealing and purification of the designed sequence, obtain the interference sequence fragment, and connect it to the RNAi lentiviral interference system construction vector by DNA ligase ( Figure 4 ).

[0066] 2. Preparation of lentiviral stock solution

[0067] Inoculate KEK293T cells in a 10 cm culture dish, and start transfection when the cells are fully spread and the confluence is about 60-70%. Add 1 mL of base medium to each 15 mL centrifuge tube, add 4 μg psPAX2, 2 μg pMD2.G, and 5 μg of target plasmid to each 1 mL of base medium, and add 30 μL of LipoFiter to each 1 mL of base medium. Then incubate at room temperature for 5 min, mix the two solutions, incubate at room temperature for 15 min, invert once for 3-5 min, replace HEK293T cells with 5 mL of complete medium, add the solution in the above centrifuge tube and mix, and replace it with 10 mL of complete medium after 6 hours. Collect the viral supernatant in the culture dish 48 h and 72 h after transfection, and filter through a 0.45 μm filter membrane to remove cell debris (it can be stored at 4°C for a short time and stored in a -80°C refrigerator for a long time. Do not freeze and thaw repeatedly).

[0068] 3. Lentiviral infection of target cells

[0069] Inoculate the target cells to be infected in a 6-well plate, and when the cells are fully spread, the confluence is about 60-70%, and the cells are in good condition, start to transfect the cells with the virus; add the dyeing reagent Polybrene (10μg / mL) and gently mix on the workbench; after 12-16 hours of infection, change the medium and continue to culture, while observing whether the cell state is abnormal, observe the fluorescence under an inverted fluorescence microscope and take pictures; after 72-96 hours of infection, perform drug screening on the infected cells to collect more successfully infected cells.

[0070] 3. Screening of stable transgenic strains

[0071] The cells were diluted into 10 96-well plates by limiting dilution method. The growth of monoclonal cells was observed after one week, and the grown monoclonal cells were transferred to 48-well plates for expansion culture after about two weeks. The grown monoclonal cells were transferred to 24-well plates and 12-well plates for expansion culture in turn. When each monoclonal cell was expanded to 2 12-well plates, cells from one well were taken out, lysed and protein was extracted, and the knockdown monoclonal cells were detected by Western blot.

[0072] The results are as follows Figure 5As shown, compared with the control group, the relative protein expression of MSANTD2 in cells treated with the RNAi interference system was significantly reduced, indicating that the expression of MSANTD2 protein in DLD1 cells and SW1116 cells was effectively inhibited.

[0073] 4. Cell proliferation assay (CCK-8 assay)

[0074] Digest and collect cells from each group 48 hours after transfection, centrifuge and set aside; resuspend cells in complete medium and adjust cell density to 30,000 cells / mL; add 100 μL of cell suspension to each well, set 5 duplicate wells for each group, and gently tap the 96-well plate to make cells evenly distributed; after cells adhere to the wall (about 6-8 hours), add CCK-8 reagent (10 μL per well) at 0, 24, 48, 72, and 96 hours, respectively, and gently tap the 96-well plate, put it in the incubator for 2 hours, then take it out and detect the absorbance value at 450 nm on the microplate reader, paying attention to the linear range of the microplate reader; use Graphpadprism to statistically process the measured data and draw a line graph.

[0075] The results are as follows Figure 6 As shown, after knocking down MSANTD2 by RNAi interference system, the proliferation activity of DLD1 cells and SW1116 cells decreased.

[0076] 5. Cell invasion and migration assay (transwell chamber method)

[0077] Digest and collect cells from each group 48 hours after transfection, centrifuge and set aside; resuspend the cells in RPMI-1640 medium and adjust the cell density to 5×10 4 / mL; add 800 μL of complete medium to a 24-well plate, place in a small chamber, fully infiltrate, take 100 μL of cell suspension and add to the upper chamber of the transwell chamber (matrigel needs to be laid in advance for invasion experiments); take out after routine culture for 24-48 hours, wash twice with 1×PBS, fix with 4% paraformaldehyde for 20 minutes, and wash twice with 1×PBS; add 500 μL of crystal violet staining solution to a 24-well plate, place in a small chamber, take out after 10 minutes, wash twice with 1×PBS, invert the small chamber, and gently wipe off the cells in the upper chamber that have not passed through with a cotton swab;

[0078] The results are as follows Figure 7 As shown in Figure 2, the invasion and migration abilities of DLD1 cells and SW1116 cells were decreased after knocking down MSANTD2 by RNAi interference system ( Figure 7 A).

Claims

1. MSANTD2 The invention relates to an application of a RNAi lentiviral interference system for the preparation of a drug for treating colorectal cancer, characterized in that: Said MSANTD2 The sequence of the RNAi lentiviral interference system of the gene is as follows: MSANTD2 -shRNA1:5'-TTTGAGCAAGCAAGATATATA-3', MSANTD2 -shRNA2:5’-ACTCTACAGCAGTGCTTATTT-3’。 2. The use according to claim 1, characterized in that: The MSANTD2 RNAi lentiviral interference system MSANTD2 Gene target inhibition in colorectal cancer cells MSANTD2 Protein expression.

3. The use according to claim 1, characterized in that: The MSANTD2 RNAi lentiviral interference system MSANTD2 Gene knockout in colorectal cancer cells MSANTD2 Gene.

4. The use according to claim 1, characterized in that: The MSANTD2 RNAi lentiviral interference system MSANTD2 Gene silencing as a target in colorectal cancer cells MSANTD2 Gene.

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