TMEM115 gene and RNAi lentivirus interference system and application thereof

Abstract

The invention discloses a TMEM115 gene as well as an RNAi lentivirus interference system and application thereof, and relates to the technical field of biological medicines. The application of the TMEM115 gene in preparation of a medicine for treating cancer or a kit for diagnosis / treatment / prognosis judgment of liver cancer provides a new strategy and direction for treatment of liver cancer, has extremely high clinical application value, and is expected to bring a better treatment effect for liver cancer patients. As a target spot for precise treatment of liver cancer, the RNAi lentivirus interference system of the TMEM115 gene is specifically targeted aiming at the sequence of the TMEM115 gene, and the RNAi interference system of the TMEM115 gene is constructed. Results show that after the TMEM115 is knocked down by an RNAi lentivirus interference system, the proliferation activity of liver cancer cells is reduced, the migration ability is reduced, and the invasion ability is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and more specifically, to the TMEM115 gene (gene ID:

[0002] 11070, protein ID: Q12893) and its RNAi lentiviral interference system and application. Background Art

[0003] According to the latest statistics from GLOBOCAN, hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, with approximately 900,000 new patients each year, ranking sixth among malignant tumors, and more than 830,000 deaths from HCC, ranking second among malignant tumors. At present, the incidence of liver cancer ranks fourth among malignant tumors in my country, and the mortality rate ranks second. It has become the second largest cause of cancer death in my country, seriously threatening people's lives and health. As a solid tumor, liver cancer has the characteristics of high malignancy, insensitivity to radiotherapy, chemotherapy and immunotherapy. HCC has an insidious onset and is often diagnosed in the middle and late stages, missing the best treatment period. In addition, the 5-year overall survival rate of HCC is low, which imposes a heavy economic burden and mental pressure on patients and their families. Since the prognosis of HCC is poor at this stage and there is a lack of effective treatment methods in clinical practice, it is of great significance to explore tumor differential genes or proteins related to the clinical significance of HCC to provide potential therapeutic targets and explore new treatment strategies.

[0004] The transmembrane (TMEM) protein family consists of proteins with one or more transmembrane domains. In the life activities of cells, members of the TMEM protein family play a vital role and are widely involved in various cellular processes, including signal transduction, ion transport, and cell adhesion. As a member of this protein family, TMEM115 is located in the Golgi apparatus. The Golgi apparatus has many important functions in the cell, and TMEM115 is involved in important cellular functions such as membrane transport and vesicle transport. Recent studies have shown that TMEM115 is upregulated in lung cancer and breast cancer cells, indicating that TMEM115 may have a potential role in the development of cancer. According to existing literature, there has been no clear report on the expression of TMEM115 in HCC, its biological function, and the relationship between clinical pathological parameters. Summary of the invention

[0005] 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 TMEM115 gene. Another technical problem to be solved by the present invention is to provide an application of the RNAi lentiviral interference system of the TMEM115 gene for preparing a drug for treating liver cancer.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] Application of TMEM115 gene in preparing medicine for treating cancer.

[0008] The biomarker used for diagnosis, treatment or prognosis of liver cancer is the TMEM115 gene.

[0009] A kit for diagnosing, treating or predicting the prognosis of liver cancer contains a TMEM115 gene sequence.

[0010] Application of TMEM115 gene in the preparation of a drug for inhibiting the proliferation activity of liver cancer cells.

[0011] Application of TMEM115 gene in the preparation of drugs for inhibiting the migration ability of liver cancer cells.

[0012] Application of TMEM115 gene in the preparation of drugs for inhibiting the invasive ability of liver cancer cells.

[0013] Application of the RNAi lentiviral interference system of the TMEM115 gene in the preparation of a drug for treating liver cancer, wherein the sequence of the RNAi lentiviral interference system of the TMEM115 gene is as follows:

[0014] TMEM115-shRNA1: 5'-CCAACTCCAGACAACCACTAT-3',

[0015] TMEM115-shRNA2: 5'-GAAGGTAAAGATATGCCAGAA-3'.

[0016] The RNAi lentiviral interference system of the TMEM115 gene uses the TMEM115 gene as a target to inhibit the expression of TMEM115 protein in liver cancer cells.

[0017] The TMEM115 gene RNAi lentiviral interference system uses the TMEM115 gene as a target to knock out the TMEM115 gene in liver cancer cells.

[0018] The TMEM115 gene RNAi lentiviral interference system uses the TMEM115 gene as a target to silence the TMEM115 gene in liver cancer cells.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1) The TMEM115 gene / protein disclosed in the present invention is a target for precision treatment of liver cancer, and is used to prepare drugs for treating liver cancer and kits for diagnosis / treatment / prognosis of liver cancer. It provides a new strategy and direction for the treatment of liver cancer, has extremely high clinical application value, and is expected to bring better treatment effects to liver cancer patients.

[0021] 2) The present invention targets the TMEM115 gene sequence, specifically targets the TMEM115 gene RNAi lentiviral interference system, and constructs the TMEM115 gene RNAi interference system: TMEM115-shRNA1: 5'-CCAACTCCAGACAACCACTAT-3', TMEM115-shRNA2: 5'-GAAGGTAAAGATATGCCAGAA-3'. After knocking down TMEM115 by the RNAi lentiviral interference system, the proliferation activity, migration ability, and invasion ability of liver cancer cells decreased. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The results of immunofluorescence detection of TMEM115 protein expression in liver cancer and adjacent tissues (A is the immunofluorescence staining of TMEM115 in liver cancer tissues; B is the immunofluorescence staining of TMEM115 in adjacent tissues);

[0023] Figure 2 This is a graph showing the relationship between high and low expression of TMEM115 and the prognosis of liver cancer patients;

[0024] Figure 3 The expression of TMEM115 in Huh-7 cells, Hep3B cells, SNU-182 cells and PLC / PRF / 5 cells was detected by Western Blot;

[0025] Figure 4 The plasmid diagram of the lentiviral knockdown vector (A) and the protein expression diagram after knocking down the TMEM115 gene in Huh-7 cells (B);

[0026] Figure 5 This is a graph showing the proliferation activity of Huh-7 cells detected by CCK-8C after knocking down TMEM115;

[0027] Figure 6 The migration ability (A) and invasion ability (B) of Huh-7 cells after knockdown of TMEM115. DETAILED DESCRIPTION

[0028] 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.

[0029] The main reagents and consumables used in the following examples are:

[0030] Multiplex immunofluorescence kit: BioNano Panorama Biotechnology Co., Ltd.

[0031] Rabbit anti-human TMEM115 antibody: NBP1-80898, Novus Corporation.

[0032] Rabbit anti-human Cytokeratin (CK) antibody: 26411-1-AP, Proteintech.

[0033] Prestained protein marker: Nanjing Novozymes Biotech Co., Ltd.

[0034] SDS-PAGE gel preparation kit: Shanghai Yazyme Biopharmaceutical Technology Co., Ltd.

[0035] Hepatocellular carcinoma cell lines (Huh-7, Hep3B, SNU-182 and PLC / PRF / 5) and HEK293T cell lines: Nanjing Kebai Biotechnology Co., Ltd.

[0036] DMEM medium, 1640 medium and fetal bovine serum: Gibco, USA.

[0037] Polybrene: Beijing Solebren Technology Co., Ltd.

[0038] Puromycin: Beijing Solebow Technology Co., Ltd.

[0039] PEI 40K transfection reagent: Wuhan Saiweier Biotechnology Co., Ltd.

[0040] 0.45μm filter: White Shark Biotechnology Co., Ltd.

[0041] PVDF membrane (for Western blot experiments): Millipore, USA.

[0042] ECL developer: Suzhou Xinsaimei Company.

[0043] Cell freezing medium: Suzhou Xinsaimei Company.

[0044] Cell Counting Kit-8 (CCK-8) detection kit: Dalian Meilun Biotechnology Co., Ltd.

[0045] Transwell chamber: Corning Company, USA.

[0046] The main instruments used in the following examples are as follows:

[0047] Tissue chip production instrument: Beecher Instruments, USA.

[0048] Inverted phase contrast microscope: Olympus, Japan.

[0049] Gel imaging system: China Tianneng Company.

[0050] Multifunctional microplate reader: Thermo Corporation, USA.

[0051] Multispectral pathology scanning system: Perkin Elmer, USA.

[0052] The liver cancer paraffin tissue chips used in the following examples were stored in the Clinical Resource Translation Research Center of the Affiliated Hospital of Nantong University (surgery was performed between 2010 and 2017, the patients had not received immunotherapy, chemotherapy or radiotherapy before surgery, and the clinical case data were detailed and complete), including 153 cases of liver cancer tissue and 25 cases of adjacent cancer tissue.

[0053] Example 1

[0054] 1. Immunofluorescence staining

[0055] The tissue chip was placed on a drying machine for baking and drying, respectively, for 1 hour each; then dewaxing, hydration and microwave repair were performed. The tissue range was drawn on the tissue chip with an immunohistochemistry pen, and the primary antibody blocking solution was added for 10 minutes, followed by the addition of the primary antibody and incubation at 4°C overnight. The next day, the tissue was reheated at room temperature for 30 minutes, the primary antibody was removed, and the tissue was rinsed with PBS 3 times, 5 minutes each time, and the secondary antibody was added and incubated at room temperature for 10 minutes, then washed with PBS 3 times, 5 minutes each time, and the dye was added for staining, incubated at room temperature for 10 minutes, and finally repaired and stained again in a cycle until the target antibody was labeled. DAPI staining was performed to observe the cell nucleus, and a coverslip was installed.

[0056] The tissue microarray was scanned by the Vectra automatic quantitative pathology imaging scanning system (3.0, PerkinElmer, USA). The staining of TMEM115 was then statistically analyzed by inForm image analysis software (2.4.1, PerkinElmer, USA). Based on the 5-year survival of the patients, the TMEM115 expression-related data were converted into binary data (low and high) using X-tile software, with 0-43 as low expression and 43-100 as high expression. All data were processed using SPSS statistical software, and the chi-square test was used for inter-group comparisons. The Cox proportional hazard regression was used to analyze the prognostic factors of the patients. The Kaplan-Meier method and log-rank test were used for univariate analysis of the prognosis of the patients. All test results were considered statistically significant at P<0.05.

[0057] The results are as follows Figure 1 and Figure 2 As shown, the expression of TMEM115 protein in liver cancer tissue is higher than that in adjacent tissues, and patients with high expression of TMEM115 have a short survival time and poor prognosis.

[0058] Example 2

[0059] 1. Culture of Hepatoma Cell Lines

[0060] Huh-7 cells and Hep3B cells were cultured with DMEM complete medium, and SNU-182 cells and PLC / PRF / 5 cells were cultured with RPMI-1640 complete medium. The temperature in the cell culture incubator was maintained at 37°C, CO2 concentration was 5%, and relative saturated humidity was 95%. For routine culture, the medium was changed or subcultured every 1-2 days, and cells in the logarithmic growth phase were selected for experiments.

[0061] 2. Extraction of total cell protein

[0062] Collect liver cancer cells in the logarithmic growth phase, discard the culture medium, and wash the cells twice with pre-cooled PBS; add different volumes of cell lysis buffer according to the specifications of the cell culture bottle, then scrape the cells with a cell scraper and transfer them to a clean EP tube. Place the scraped cell protein on ice to fully lyse for 30 minutes. Centrifuge at 4℃ for 10 minutes, obtain the supernatant, and use a UV spectrophotometer to determine the concentration of cell protein. Then add 5× loading buffer and blow and mix evenly, boil at 95℃ for 10 minutes, and store in a -20℃ refrigerator for later use.

[0063] 3. Western blot

[0064] Prepare polyacrylamide gel (5% concentrated gel, 10% separation gel); after loading the protein marker and the extracted protein sample, adjust the operating voltage to 80V, and adjust the voltage to 120V after 30min. After the protein is separated, take out the gel and transfer to the membrane (PVDF membrane); transfer at a constant current of 400mA for 30min; after the transfer, put the PVDF membrane into the blocking solution and block it at room temperature for 2h; dilute the antibody with the primary antibody diluent, and incubate the PVDF membrane in the primary antibody at 4℃ overnight; after washing the membrane, prepare the secondary antibody diluent with TBST, and incubate the PVDF membrane in the secondary antibody at room temperature for 1h; after washing the membrane again, spread the PVDF membrane in the developer, evenly add the ECL luminescent solution on the PVDF membrane, and take pictures and save it with the gel imaging system.

[0065] The results are as follows Figure 3 As shown, TMEM115 was most highly expressed in Huh-7 hepatoma cells.

[0066] Example 3

[0067] 1. Construction of RNAi lentiviral interference system of TMEM115 gene

[0068] Aiming at the sequence of TMEM115 gene (gene ID: 11070, protein ID: Q12893), an RNAi lentiviral interference system specifically targeting the TMEM115 gene was constructed to construct a lentiviral-mediated RNAi interference system. The gene sequence of the RNAi interference system of the TMEM115 gene is as follows:

[0069] TMEM115-shRNA1: 5'-CCAACTCCAGACAACCACTAT-3',

[0070] TMEM115-shRNA2: 5'-GAAGGTAAAGATATGCCAGAA-3'.

[0071] 2. Preparation of lentiviral stock solution

[0072] HEK293T cells were seeded into a 100mm cell culture dish, and transfection was started when the confluence was 60-70%; 500μL basal culture medium, 5μg target plasmid, 4μg psPAX2, and 2μg pMD2.G were added to 1.5mL EP tubes; 500μL basal culture medium and 30μL PEI 40K transfection reagent were added to 1.5mL EP tubes; incubated at room temperature for 5min; the two solutions were mixed evenly and incubated at room temperature for 15min; HEK293T cells were replaced with 5mL complete culture medium, and the above 1mL solution was added and mixed; fresh culture medium was replaced 6h after transfection, and the virus supernatant was collected 48h and 72h after transfection, respectively, and then filtered using a 0.45μm filter, and stored in a -80℃ refrigerator after aliquoting.

[0073] 3. Lentiviral infection of target cells

[0074] The target cells were inoculated into a 6-well plate, and the cell concentration was selected to be 30-50% for infection according to the cell growth rate; an appropriate volume of virus was diluted into 1 mL of complete culture medium, and Polybrene was added to promote lentiviral infection so that the final concentration in the solution was 5 μg / mL; the culture medium in the six-well plate was discarded, PBS was added to wash away the residual culture medium, and then the complete culture medium containing the virus and Polybrene was added to the six-well plate, and the plate was placed in an incubator for culture; after 24 hours of infection, the virus infection solution was discarded, the cell culture medium was added, and the cell status was observed at the same time.

[0075] 4. Screening of stable cell lines

[0076] 96 hours after infection, the fluorescence was observed under an inverted fluorescence microscope. In order to screen the cell lines stably expressing the infected virus, 1 μg / mL Puromycin was added to the culture medium for 3-5 days to kill the uninfected cells, and the successfully infected monoclonal cells were collected and digested and transferred to a 96-well plate; the growth of the monoclonal cells was observed after one week, and the grown monoclonal cells were transferred to a 48-well plate for expansion and culture after about two weeks; the grown monoclonal cells were transferred to a 24-well plate and a 12-well plate for expansion and culture in turn; when each monoclonal clone was expanded to 2 12-well plates, the cells in one well were taken out, the protein was lysed and extracted, and the monoclonal strains with gene knockdown were detected by Western blot.

[0077] The results are as follows Figure 4 As shown, compared with the control group, the relative protein expression of TMEM115 in cells treated with the RNAi lentiviral interference system was significantly reduced, indicating that the expression of TMEM115 protein in Huh-7 cells was effectively inhibited.

[0078] 5. Cell proliferation assay (CCK-8 assay)

[0079] Digest and collect cells from each group (shNC group, TMEM115-sh1 group, and TMEM115-sh2 group), adjust the cell amount, add 2000 cells per well to a 96-well plate, and set 4 replicates for each group; after the cells adhere to the wall (about 6-8h), set different time points (0, 24, 48, 72, 96h). Discard the original culture medium, use the basal culture medium and CCK-8 detection reagent in a ratio of 9:1 to prepare the detection reagent, add 100μL to each empty space, and incubate at 37℃ for 2h. Detect the absorbance value at 450nm on an ELISA reader, use Graphpadprism to statistically analyze the measured data, and draw a line graph.

[0080] The results are as follows Figure 5As shown, the proliferation activity of Huh-7 cells decreased after knocking down TMEM115 using the RNAi lentiviral interference system.

[0081] 6. Cell migration assay (Transwell chamber method)

[0082] The cells of each group (shNC group, TMEM115-sh1 group and TMEM115-sh2 group) were digested and collected, and the cells were resuspended in medium to adjust the cell density to 4×10 5 / mL; add 800 μL of complete culture medium to a 24-well plate, put it into a small chamber, fully infiltrate, take 200 μL of cell suspension and add it to the upper chamber of the Transwell chamber; take it out after routine culture for 24 hours, wash it twice with 1×PBS, fix it with 4% paraformaldehyde for 20 minutes, and wash it twice with 1×PBS; add 500 μL of crystal violet staining solution to a 24-well plate, put it into a small chamber, take it out after 10 minutes, wash it twice with 1×PBS, invert the small chamber, gently wipe off the cells in the upper chamber that have not passed through with a cotton swab, and observe the results with an inverted microscope.

[0083] The results are as follows Figure 6 As shown in A, the migration ability of Huh-7 cells was significantly decreased after knocking down TMEM115 by RNAi lentiviral interference system.

[0084] 7. Cell invasion assay (Transwell chamber method)

[0085] (1) Prepare the hydrogel (mix 50 μL of hydrogel in 350 μL of diluent, then add 50 μL of base medium to mix), add 80 μL to the upper chamber of the Transwell chamber, and avoid bubbles; (2) Digest and collect cells from each group that have been successfully infected with lentivirus, centrifuge and set aside; (3) Resuspend the cells in base medium and adjust the cell density to 8 × 10 5 / mL; (4) Add 800 μL of complete culture medium to a 24-well plate, place the chamber, fully infiltrate, and take 200 μL of the cell suspension to the upper chamber of the Transwell chamber; (5) After routine culture for 24-48 h, remove the plate, wash twice with 1× PBS, fix with 4% paraformaldehyde for 20 min, and wash twice with 1× PBS; (6) Add 500 μL of crystal violet stain to a 24-well plate, place the chamber, remove after 10 min, wash twice with 1× PBS, invert the chamber, gently wipe off the cells in the upper chamber that have not passed through with a cotton swab, and observe the results with an inverted microscope.

[0086] The results are as follows Figure 6 As shown in B, the invasion ability of Huh-7 cells was significantly decreased after knocking down TMEM115 by RNAi lentiviral interference system.

Claims

1. Application of TMEM115 gene in the preparation of drugs for treating cancer.

2. A biomarker for diagnosis, treatment or prognosis of liver cancer, characterized in that: It is the TMEM115 gene.

3. A kit for diagnosis, treatment or prognosis of liver cancer, characterized in that: Contains TMEM115 gene sequence.

4. Application of TMEM115 gene in the preparation of drugs for inhibiting the proliferation activity of liver cancer cells.

5. Application of TMEM115 gene in the preparation of drugs for inhibiting the migration ability of liver cancer cells.

6. Application of TMEM115 gene in the preparation of drugs for inhibiting the invasive ability of liver cancer cells.

7. Application of the RNAi lentiviral interference system of the TMEM115 gene in the preparation of a drug for treating liver cancer, characterized in that: The sequence of the RNAi lentiviral interference system of the TMEM115 gene is as follows: TMEM115-shRNA1: 5'-CCAACTCCAGACAACCACTAT-3', TMEM115-shRNA2: 5'-GAAGGTAAAGATATGCCAGAA-3'.

8. The use according to claim 7, characterized in that: The RNAi lentiviral interference system of the TMEM115 gene uses the TMEM115 gene as a target to inhibit the expression of TMEM115 protein in liver cancer cells.

9. The use according to claim 7, characterized in that: The TMEM115 gene RNAi lentiviral interference system uses the TMEM115 gene as a target to knock out the TMEM115 gene in liver cancer cells.

10. The use according to claim 7, characterized in that: The TMEM115 gene RNAi lentiviral interference system uses the TMEM115 gene as a target to silence the TMEM115 gene in liver cancer cells.

Images