Application of inhibiting substance of E3 ubiquitin ligase SYVN1 in preparation of medicine for treating gastric cancer

Abstract

According to the application of the inhibiting substance of the E3 ubiquitin ligase SYVN1 in preparation of the gastric cancer treatment medicine, the expression level of the target SYVN1 targeted by the adopted small interfering RNA in cancer tissue of a gastric cancer patient is higher than that in para-carcinoma tissue, the expression level is in negative correlation with survival time, and the unique clinical application value is achieved.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and relates to the application of an inhibitory substance of E3 ubiquitin ligase SYVN1 in the preparation of a drug for treating gastric cancer. Background Art

[0002] Gastric cancer (GC) is the fifth most common cancer and the third most common cause of cancer-related death in the world, with approximately 800,000 deaths worldwide. China is a high-incidence area for gastric cancer, accounting for nearly 50% of the total cases of gastric cancer worldwide.

[0003] The current treatments for gastric cancer include surgical treatment, chemotherapy, radiotherapy, endoscopic treatment, etc., but each treatment method still has some limitations. Especially for drug treatment, although a large number of drugs for treating gastric cancer have been used in clinical practice, they have large side effects, are prone to drug resistance, and individual differences lead to limited efficacy. Therefore, it is of great significance to discover drug targets for the treatment of gastric cancer.

[0004] SYVN1 (HMG-CoA reductase degradation protein 1), located on the endoplasmic reticulum membrane, is also known as HRD1. SYVN1 is an E3 ubiquitin ligase that can participate in the endoplasmic reticulum-associated degradation pathway (ERAD). Its main function is to mediate the degradation of important proteins in the body, thereby participating in the regulation of protein synthesis and transcription factor regulation. Recent studies have revealed that SYVN1 plays a very important role in a variety of malignant tumors.

[0005] Small interfering RNA (siRNA) is a double-stranded RNA molecule that interferes with the expression of specific genes and is mainly involved in the RNA interference (RNAi) mechanism in cells. By introducing synthetic siRNA or using vectors expressing siRNA, a gene can be specifically silenced to help study its biological function. In addition, siRNA can target cancer-related oncogenes, inhibit cancer cell proliferation, angiogenesis, or induce cancer cell apoptosis. siRNA drugs such as Patisiran are the first FDA-approved siRNA drugs for the treatment of hereditary ATTR amyloidosis (hATTR). The above makes it possible to use some molecules that are closely related to the occurrence and development of tumors as specific targets, and silence them through RNAi technology to achieve anti-tumor purposes. At present, this technology has been widely used in the field of gene therapy for malignant tumors. Summary of the invention

[0006] The purpose of the present invention is to provide an application of an inhibitory substance of E3 ubiquitin ligase SYVN1 in the preparation of a drug for treating gastric cancer.

[0007] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: use of an inhibitory substance of E3 ubiquitin ligase SYVN1 in the preparation of a drug for treating gastric cancer.

[0008] The preferred technical solution is: the inhibitory substance is a small interfering RNA that downregulates the expression of the SYVN1 gene in gastric cancer cells.

[0009] The preferred technical solution is: the nucleotide sequence of the 5'-3' direction of the sense strand of the small interfering RNA is:

[0010] 5'-GCAUUGUCUCUCUUAUGUUZZ-3'Z=dT;

[0011] The nucleotide sequence of the antisense strand in the 5'-3' direction is:

[0012] 5'-AACAUAAGAGAGACAAUGCZZ-3', where Z=dT.

[0013] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: use of a recombinant vector in the preparation of a drug for treating gastric cancer, wherein the recombinant vector contains small interfering RNA.

[0014] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0015] 1. The target SYVN1 targeted by the small interfering RNA of the present invention is highly expressed in gastric cancer tissues, and downregulating SYVN1 by small interfering RNA technology can significantly inhibit the proliferation, migration, invasion and cloning ability of gastric cancer cells. The technical method adopted by the present invention can provide a specific drug for the treatment of gastric cancer, which can help solve the problems of non-surgical treatment effect and poor prognosis of advanced gastric cancer, and has important application value.

[0016] 2. The present invention provides a new treatment method for gastric cancer, which provides an effective treatment method for patients with advanced gastric cancer, and solves the technical problems of poor efficacy and poor prognosis of existing treatment methods for advanced gastric cancer;

[0017] 3. The small interfering RNA in the present invention can inhibit the proliferation, migration, invasion and cloning ability of gastric cancer cells, and inhibit the occurrence and development of gastric cancer;

[0018] 3. The target SYVN1 targeted by the small interfering RNA used in the present invention has a higher expression level in the cancer tissue of gastric cancer patients than in the adjacent tissues, and is negatively correlated with the survival time, and has unique clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : AB figures are immunohistochemical experiments to detect the expression of SYVN1 in normal gastric tissue and gastric cancer tissue. The results show that the expression level of SYVN1 in gastric cancer tissue is significantly higher than that in normal tissue; CD figures are immunohistochemical experiments to detect the expression of SYVN1 in gastric cancer tissue and adjacent cancer tissue. The results show that the expression level of SYVN1 in gastric cancer tissue is significantly higher than that in adjacent cancer tissue; E figures are immunoblotting to detect the expression of SYVN1 in gastric cancer tissue and adjacent cancer tissue. The primary antibodies in the figures are SYVN1 antibody and GAPDH antibody. The results also show that the expression level of SYVN1 in gastric cancer tissue is significantly higher than that in adjacent cancer tissue.

[0020] Figure 2 :Using the survival time samples in the TCGA database, the correlation between SYVN1 gene expression and overall survival time was analyzed. The results showed that with the increase of SYVN1 expression, the overall survival time of cancer patients was significantly shortened.

[0021] Figure 3 : Figure A shows the protein expression of SYVN1 in five gastric cancer cell lines detected by immunoblotting, and the primary antibodies in the upper figure are SYVN1 antibody and GAPDH antibody; Figure B shows the mRNA expression of SYVN1 in five gastric cancer cell lines detected by RT-qPCR; Figures C and D show the efficiency of overexpressing SYVN1 in MGC803 and AGS cells detected by immunoblotting, and the primary antibodies in the figures are SYVN1 antibody and GAPDH antibody; Figures E and F show the efficiency of knocking down SYVN1 in HCG-27 and SGC7901 cells detected by immunoblotting, and the primary antibodies in the figures are SYVN1 antibody and GAPDH antibody; Figures G and H show the mRNA expression level of SYVN1 after overexpressing SYVN1 in MGC803 and AGS cells detected by RT-qPCR, and Figures I and J show the mRNA expression level of SYVN1 after knocking down SYVN1 in HCG-27 and SGC7901 cells detected by RT-qPCR. Above, CJ graphs detected the knockdown efficiency of SYVN1 small interfering RNA in gastric cancer cells.

[0022] Figure 4: Figures A and B show CCK-8 experiments to detect that overexpression of SYVN1 promoted the proliferation ability of MGC803 and AGS cells, while knockdown of SYVN1 inhibited the proliferation ability of HCG-27 and SGC7901 cells; Figures C and D show cell soft agar cloning experiments to detect that overexpression of SYVN1 promoted the cloning ability of MGC803 and AGS cells, while knockdown of SYVN1 inhibited the cloning ability of HCG-27 and SGC7901 cells.

[0023] Figure 5 : Figures A and B show scratch assays to detect that overexpression of SYVN1 promoted the migration ability of MGC803 and AGS cells, while knockdown of SYVN1 inhibited the migration ability of HCG-27 and SGC7901 cells; Figures C and D show Transwell assays to detect that overexpression of SYVN1 promoted the invasion ability of MGC803 and AGS cells, while knockdown of SYVN1 inhibited the invasion ability of HCG-27 and SGC7901 cells. DETAILED DESCRIPTION

[0024] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in the embodiments.

[0025] See also Figure 1-5 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions under which the present invention can be implemented. Therefore, they have no substantive technical significance, and any structural modification, change in proportional relationship or adjustment of size. The following examples are provided to better understand the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0026] The invention discloses an inhibitory substance for E3 ubiquitin ligase SYVN1, which is a small interfering RNA that downregulates the SYVN1 gene in gastric cancer cells. The gastric cancer treatment drug of the present invention comprises an inhibitory substance for SYVN1, which can solve the problems of poor efficacy and poor prognosis of traditional gastric cancer treatment methods.

[0027] In gastric cancer patients, the expression level of SYVN1 in cancer tissue is higher than that in normal tissue and adjacent tissue, and is negatively correlated with survival time; knocking down SYVN1 with small interfering RNA can inhibit the proliferation, migration, invasion and cloning ability of gastric cancer cells.

[0028] Use of an agent for inhibiting or downregulating the expression of the E3 ubiquitin ligase SYVN1 gene in the preparation of a drug for treating gastric cancer.

[0029] The reagent for inhibiting or down-regulating the expression of the E3 ubiquitin ligase SYVN1 gene is a small interfering RNA (siRNA) targeting the E3 ubiquitin ligase SYVN1 gene, which can play a role in continuously inhibiting or down-regulating the expression of the target gene in cells. The positive chain nucleotide sequence of the small interfering RNA is shown in SEQ ID NO.1; the antisense chain nucleotide sequence is shown in SEQ ID NO.2.

[0030] Justice Chain:

[0031] GCAUUGUCUCUCUUAUGUU(dT)(dT)(SEQ ID NO.1);

[0032] 5'-GCAUUGUCUCUCUUAUGUUZZ-3'Z=dT;

[0033] Antisense strand:

[0034] AACAUAAGAGAGACAAUGC(dT)(dT)(SEQ ID NO.2);

[0035] 5'-AACAUAAGAGAGACAAUGCZZ-3', Z in the sequence = dT

[0036] The gastric cancer therapeutic drug can inhibit the proliferation, migration, invasion and cloning ability of gastric cancer cells, and can inhibit the occurrence and development of gastric cancer cells.

[0037] Three small interfering RNA sequences were screened in the early stage, and only the above sequence had the best effect. The small interfering RNA fragment targeting the SYVN1 gene was synthesized by Shanghai Sangon Biotechnology Co., Ltd. and was confirmed to be an effective sequence that can downregulate the expression of SYVN1 in gastric cancer cells.

[0038] The SYVN1 small interfering RNA oligonucleotide sequence is a lyophilized powder, stored at -20°C, and dissolved in DEPC water before use.

[0039] The principle of the present invention is: small interfering RNA is transfected into cells using a transfection reagent, and the small interfering RNA is unzipped into a sense strand and an antisense strand under the action of the cell RNA helicase; the antisense strand is recruited by the RNA-induced silencing complex (RISC) and integrated into RISC; the antisense strand of the small interfering RNA binds to the target mRNA through the principle of base complementarity to form a double-stranded RNA (dsRNA), and this binding recruits a nuclease to cut the target mRNA into fragments, thereby preventing it from being translated into protein, resulting in downregulation of gene expression.

[0040] A use of a recombinant vector in the preparation of a drug for treating gastric cancer, wherein the recombinant vector comprises the above-mentioned small interfering RNA targeting the E3 ubiquitin ligase SYVN1 gene.

[0041] A drug for treating gastric cancer, comprising the above-mentioned small interfering RNA targeting the E3 ubiquitin ligase SYVN1 gene.

[0042] Example 1: E3 ubiquitin ligase SYVN1 is a target for the treatment of gastric cancer

[0043] Experimental materials involved in the experiment:

[0044] 1. Cell lines: Human gastric cancer cells MGC803, AGS, HCG-27 and SGC7901 were obtained from the Cell Bank of the Chinese Academy of Sciences.

[0045] 2. Experimental Materials, Reagents and Kits

[0046] DMEM culture medium: Wuhan Sewell Biotechnology Co., Ltd.; fetal bovine serum: Thermo Fisher Scientific; RIPA lysis buffer: Shanghai Bioyuntian Biotechnology Co., Ltd.; PMSF: Shanghai Bioyuntian Biotechnology Co., Ltd.; protease inhibitor cocktail: Shanghai Bioyuntian Biotechnology Co., Ltd.; ECL color development solution: Wuhan Sewell Biotechnology Co., Ltd.; SDS-PAGE gel preparation kit: Shanghai Bioyuntian Biotechnology Co., Ltd.; PVDF membrane: Millipore; transfection reagent: Thermo Fisher Scientific; anhydrous ethanol: Sinopharm Chemical Reagent Co., Ltd.; Transwell chamber: Corning; 1× crystal violet staining solution: Shanghai Bioyuntian Biotechnology Co., Ltd.; ready-to-use immunohistochemistry detection kit: Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

[0047] 4. Antibodies: (1) SYVN1 antibody (rabbit source): purchased from proteintech, catalog number: 13473-1-AP; (2) GAPDH antibody (mouse source): purchased from proteintech, catalog number: 10494-1-AP.

[0048] 5. Main instruments and equipment

[0049] A2 biological safety cabinet: China's Bo Ke Group Co., Ltd.; BD series carbon dioxide cell culture incubator: Germany's Binder Company; 224s precision electronic balance: Germany's Sartorius Company; electronic analytical balance: China's Shaying Company; -80℃ ultra-low temperature medical refrigerator: China's Haier Company; 4℃ refrigerator: China's Haier Company; ordinary room temperature shaker: Taicang Hualida Experimental Equipment Co., Ltd.; 4℃ standard product display cabinet: Beijing Fuyi Co., Ltd.; EVOS inverted fluorescence microscope purchased from Thermo Fisher Scientific in the United States; VE180 and EPS300 vertical electrophoresis tanks and electrophoresis apparatus purchased from Shanghai Tianneng Co., Ltd.; G3E microwave oven purchased from China's Midea Company; 0-150MM high-precision vernier caliper purchased from Shanghai Menite Instrument Co., Ltd. Specific implementation method:

[0051] 1. Immunohistochemical assay showed that the expression level of SYVN1 in gastric cancer tissues was significantly higher than that in adjacent tissues ( Figure 1 C and Figure 1 as shown in D).

[0052] (1) Fixation, dehydration, embedding, sectioning (4 μm), slicing (46°C-48°C), and baking (90°C, 20 min).

[0053] (2) Dewaxing: Immerse the slide in xylene I / II and place it in a 65°C oven for 1 hour, then place it back in a fume hood and slowly cool it down. Then rinse it in different concentrations of alcohol, such as 100%, 90%, 80%, and 70%, for 5 minutes each time, and then rinse it in double-distilled deionized water for 5 minutes. Finally, rinse it with PBS solution for about 3 times, with an interval of 5 minutes each time.

[0054] (3) During the waiting period in step (2), dilute the sodium citrate antigen retrieval solution (50X) to 1X: add 10 mL of sodium citrate antigen retrieval solution (50X) to 490 mL of double-distilled deionized water, and mix a total of 500 mL of retrieval solution.

[0055] (4) Microwave repair: Immerse the slide in the prepared repair solution, heat it at high heat for 10 min, then stop for 5 min, heat it at high heat for another 5 min, and finally cool it to room temperature in a fume hood.

[0056] (5) Wash the slides with PBS buffer for about 3 times, with an interval of 5 minutes between each wash. Meanwhile, prepare 0.5% periodic acid in a dark place: add 0.1 g of periodic acid to 20 mL of double distilled deionized water and mix.

[0057] (6) Carefully wipe away the water around the tissue with paper, draw a circle with a pen, and gently add the prepared periodic acid solution. Incubate at 37°C for about 20 minutes, then wash with PBST for about 3 times, with an interval of 5 minutes between each wash.

[0058] (7) Add goat serum and block at 37°C for 30 min.

[0059] (8) During the blocking period, prepare the primary antibody at the same time to save time. The primary antibody is diluted with 0.5% Triton.

[0060] (9) Shake off the goat serum blocking solution on the slide directly by hand. Carefully add the primary antibody, place the slide in a wet box, and incubate it in a refrigerator at 4°C overnight.

[0061] (10) The next day, remove the wet box. If there is still a lot of antibody on the slide, rewarm it at 37°C for 30 min. If there is less antibody, keep it at room temperature for 30 min.

[0062] (11) Wash off the primary antibody with PBST for 5 min each time for a total of 6 times.

[0063] (12) Add secondary antibody using a three-step method: first add yellow IgG and incubate in a 37°C oven for about 30 min, then add red horseradish enzyme and incubate again at 37°C for 30 min, and finally wash with PBST for 30 min to 1 hour.

[0064] (13) Prepare DAB colorimetric solution: add 1 drop of DAB stock solution to 1 mL of DAB diluent.

[0065] (14) Carefully add DAB under a microscope. Start timing as you add the DAB. Record the time it takes for brown to appear under the microscope. Gently rinse under running water for a while, then stain with hematoxylin for 40 seconds. Finally, rinse the slide under running water until the water has no color. Observe the slide under a microscope. If the nucleus is too dark, continue to rinse with running water. If the nucleus is too light, continue to restain.

[0066] (15) After color development, place the slide in an oven at 65°C for 20 min until completely dried.

[0067] (16) After drying, place the slide in transparent xylene overnight.

[0068] (17) Finally, seal the slides with neutral gum, being careful not to create bubbles.

[0069] 2. SYVN1 expression is negatively correlated with the survival time of gastric cancer patients ( Figure 2 shown);

[0070] Gastric cancer tissue samples with complete survival time were downloaded from the TCGA database. The samples were divided into SYVN1 high expression group and low expression group based on the median SYVN1 expression level, and Kaplan-Meier survival curve analysis was performed. Figure 2 The Kaplan-Meier survival curve of gastric cancer patients is given. As shown in the figure, SYVN1 gene expression is related to overall survival time. As SYVN1 expression increases, the overall survival time of gastric cancer patients decreases significantly (P < 0.01). In the later stage, as the number of follow-up patients gradually decreases, the survival rates of gastric cancer patients in the high and low expression groups of SYVN1 tend to be similar.

[0071] 3. Overexpression of SYVN1 promotes the proliferation of gastric cancer cells, while knocking down SYVN1 inhibits the proliferation of gastric cancer cells ( Figure 4 A and Figure 4 (as shown in B).

[0072] The CCK-8 experimental method is as follows:

[0073] (1) Seed the 6-well or 24-well plates the night before.

[0074] (2) Transfection was performed on the next day, and for cells transfected with exogenous plasmids, the seed plates were digested 24-48 hours later.

[0075] (3) The trypsin-digested cells were resuspended in 1 mL of culture medium and counted three times, and the average value was calculated. About 3,000 to 5,000 cells were seeded in each well, and five 96-well plates were seeded for five time points, and then placed in a cell culture incubator for further culture.

[0076] (4) Prepare cell culture medium containing CCK-8 in a dark place on the operating table. Mix 10 μL of CCK-8 reagent in 90 μL of complete culture medium and add to each well.

[0077] (5) Gastric cancer cell lines generally adhere to the wall in about 4-6 hours. Take out a 96-well plate from the incubator and discard the old culture medium. Keep it away from light and add 100 μL of the prepared culture medium to each well. Make 3 replicates for each group. In addition, three background wells need to be set up, that is, culture medium without CCK-8 reagent, in order to reduce errors. Wrap the lid with tin foil, then cover it and place it in the incubator to continue culturing for 1-2 hours.

[0078] (6) Carefully remove the culture plate from the incubator and use a microplate reader to measure the absorbance value at A450 of each well. The value at this time is recorded as the value at 0 h. Subsequently, at four time points of 24 h, 48 h, 72 h, and 96 h, culture medium containing CCK-8 reagent is added and the same operation is performed as above.

[0079] (7) Finally, a relative proliferation curve was drawn based on the measured OD value to observe the cell proliferation rate.

[0080] 4. Overexpression of SYVN1 promotes the migration ability of gastric cancer cells, while knocking down SYVN1 inhibits the migration ability of gastric cancer cells ( Figure 5 A and Figure 5 (as shown in B).

[0081] The cell scratch assay method is as follows:

[0082] The purpose of the scratch test is to evaluate the migration ability of normal gastric cancer cell lines and gastric cancer cell lines with knockout gene and overexpression of SYVN1. First, prepare the ruler and pen needed for the experiment, place them in a sterile clean bench and sterilize them with ultraviolet light for 30 minutes. Other instruments need to be sterilized at high temperature. Then use a pen to measure the appropriate distance with a ruler on the back of the 6-well plate, and evenly draw a straight line of horizontal perforations. Each well has at least 5 parallel straight lines passing through it. Inoculate the cells into the new sterile six-well plate. When the cell growth density is between 80% and 90%, use a pipette to suck out the old culture medium in the well. Then use a 20μL yellow-tipped pipette to carefully scratch perpendicular to the 5 horizontal lines on the back, about 3-4 scratches per well. Then gently wash the six-well plate twice with PBS solution to remove dead and detached cells. Then add culture medium containing 5% serum and a small amount of double antibody to the six-well plate, and continue to culture in a 37℃, 5% CO2 incubator. The six-well plate was taken out at 0, 24, 48, 72 and 96 hours and photographed under a microscope.

[0083] 5. Overexpression of SYVN1 promotes the invasive ability of gastric cancer cells, while knocking down SYVN1 inhibits the invasive ability of gastric cancer cells ( Figure 5 C and Figure 5 as shown in D).

[0084] The Transwell assay method is as follows:

[0085] (1) Coating with basement membrane: First, dilute Matrigel and serum-free DMEM in a ratio of 1:8 on a sterile operating table, and carefully add 100 μL of the mixture to each Transwell chamber. Then place it in a sterile cell culture incubator at 37°C for 3 hours. When a thin white layer is observed at the bottom of the chamber, it means that Matrigel has polymerized into a gel. At this time, the excess liquid in the culture plate can be aspirated.

[0086] (2) Preparation of cell suspension: Digest the cells according to the above cell culture steps, collect the cell suspension and centrifuge. Discard the upper culture medium as completely as possible, then wash 2-3 times with sterile 1× PBS. Finally, resuspend the cells fully with serum-free DMEM.

[0087] (3) Inoculation of cells: First, place 500 μL of culture medium containing 20% ​​serum (appropriate amount of double antibody) in the lower well of the chamber. Then count the cells and inoculate about 100 μL of culture medium containing 4×10 4 The cell suspension of 10 cells should be placed carefully into the well, and be careful not to have bubbles between the chamber and the culture medium in the well. If bubbles are found during operation, carefully lift the chamber, remove the bubbles first, and then gently place the chamber into the culture well.

[0088] (4) After 48 hours of incubation in the incubator, fix the chamber with formaldehyde for 30 minutes at room temperature, then stain with crystal violet dye for 30-60 minutes at room temperature. Then rinse with running water, and gently wipe the excess matrix gel and crystal violet dye at the bottom of the chamber with a cotton swab. Finally, invert the chamber and observe and photograph it under a microscope.

[0089] 6. Overexpression of SYVN1 promotes the cloning ability of gastric cancer cells, while knocking down SYVN1 inhibits the cloning ability of gastric cancer cells ( Figure 4 C and Figure 4 as shown in D).

[0090] The cell soft agar cloning experiment method is as follows:

[0091] The purpose of the soft agar cell cloning experiment is to determine the malignant clone-forming ability of cells. The control cells, gastric cancer cell lines overexpressing a certain gene and knocking out the gene were suspended and replanted in a new six-well culture plate covered with agarose gel at a density of 400 cells per well. And cultured in a sterile 37°C, 5% CO2 incubator. During the culture period, the agarose gel at the bottom of the well should be frequently observed to see if it is turbid or contaminated. About 7-11 days, multiple cloned cell clusters visible to the naked eye can be seen growing at the bottom of the six-well plate, and the culture can be terminated at this time. The six-well plate was first gently washed 2-3 times with 1×PBS to remove impurities. Then the cell clusters were fixed with 4% paraformaldehyde at room temperature for 30-60 minutes. Add 1-2mL of crystal violet solution to the six-well plate and stain at room temperature for 30 minutes. After staining, the six-well plate was gently rinsed with clean water to wash off the crystal violet, and finally dried and inverted for photography.

[0092] Example 2: Establishment of a SYVN1-knockdown gastric cancer cell line using SYVN1 small interfering RNA

[0093] Experimental materials involved in the experiment:

[0094] 1. Cell lines and SYVN1 small interfering sequences:

[0095] (1) Human gastric cancer cells HCG-27 and SGC7901 were obtained from the Cell Bank of the Chinese Academy of Sciences.

[0096] (2) SYVN1 small interfering sequence was purchased from Shanghai Sangon Biotechnology Co., Ltd.

[0097] 2. Experimental Materials, Reagents and Kits

[0098] DMEM culture medium: Wuhan Sewell Biotechnology Co., Ltd.; fetal bovine serum: Thermo Fisher Scientific; transfection reagent: Shanghai Sangon Biotechnology Co., Ltd.

[0099] 3. Main instruments and equipment

[0100] A2 biological safety cabinet: China's Bo Ke Group Co., Ltd.; BD series carbon dioxide cell culture incubator: Germany's Binder Company; -80℃ ultra-low temperature medical refrigerator: China's Haier Company; 4℃ refrigerator: China's Haier Company; ordinary room temperature shaker: Taicang Hualida Experimental Equipment Co., Ltd.; 4℃ standard product display cabinet: Beijing Fuyi Co., Ltd.; EVOS inverted fluorescence microscope purchased from Thermo Fisher Scientific in the United States; VE180 and EPS300 vertical electrophoresis tanks and electrophoresis apparatus purchased from Shanghai Tianneng Co., Ltd.; centrifuge purchased from Hunan Xiangyi Centrifuge Instrument Co., Ltd. Specific implementation method:

[0102] 1. Small interfering RNA transfection

[0103] (1) Preparation of small interfering RNA: According to the dosage of synthesized siRNA, add appropriate amount of DEPC water to prepare it to a concentration of 20 nM and store it at -20°C for later use.

[0104] (2) Cell preparation: Digest and count the cells in the logarithmic growth phase and in good condition, and add an appropriate amount of cells to the culture dish so that the cell confluence in the dish reaches 80%-90% before transfection 24 hours later.

[0105] (3) Transfection process: Take a 60mm culture dish as an example. Prepare two centrifuge tubes, add 500μL of serum-free DMEN culture medium to each, add 10μL of transfection reagent to one of them, gently pipette to mix, and place at room temperature for 5 minutes; add 10uL of small interfering RNA working solution to the other. Mix the two liquids, pipette to mix, and incubate at room temperature for 20 minutes. Then, discard the culture medium in the culture dish to be transfected, add 1mL of the above mixture and 3mL of culture medium without double antibody and containing 10% fetal bovine serum, and move it into the cell culture incubator and let it stand for 24 hours.

[0106] (4) Post-transfection cell processing: 24 hours after cell transfection, discard the culture medium and transfection reagent, digest the cells and count them. Re-plate the cells for subsequent experiments based on the target.

[0107] 2. Verification of SYVN1 knockdown efficiency in gastric cancer cells: Western blot experiments were used to verify the knockdown efficiency of small interfering RNA. The Western blot experiment method is as follows:

[0108] (1) Protein sample preparation

[0109] 24 hours after the above cells are transfected or after other processing steps required in the experiment, use a pipette to gently aspirate the old culture medium in the culture dish and discard it. Then carefully wash the cells twice with a sterile 1×PBS buffer solution to remove dead cells and impurities. Then add 1-2mL of cell lysis solution containing protease inhibitors to the cell culture dish, and place the cell culture dish on ice to continue lysis at low temperature for 20-30 minutes. The purpose is to fully lyse the cells. Then mix the lysate and collect it into a 1.5mL EP tube with a pipette. At the same time, prepare boiling water at 100℃, put it in the water and continue to boil for 15 minutes to fully denature the protein.

[0110] (2) SDS-PAGE gel preparation

[0111] SDS-PAGE gel preparation

[0112]

[0113] (3) Electrophoresis

[0114] Select the size of the pre-stained protein marker according to the molecular weight of the target protein; after loading, cover the lid and turn on the power. Use 80V-90V first, and then select 120V-130V to continue electrophoresis after the pre-stained protein marker separates. The time to turn off the electrophoresis instrument is determined by the time it takes for the Loading Buffer to run to the bottom of the SDS-PAGE gel, which is generally about two hours.

[0115] (4) Transfer

[0116] After the above electrophoresis step is completed, carefully cut the SDS-PAGE gel and transfer it to the prepared transfer buffer. According to the molecular weight of the protein, transfer it in the transfer tank at low temperature for a certain period of time (note that the temperature should not be too high). When the target protein can be completely transferred to the PVDF membrane, the transfer can be completed.

[0117] (5) Detection

[0118] After the above transfer steps are completed, carefully place the PVDF membrane in an appropriate container to avoid contamination. Use a balance to weigh 1g of skim milk powder and dissolve it in a 50mL centrifuge tube containing 20mL1×PBST. Incubate the milk and PVDF membrane at room temperature for 1 hour. After the incubation, wash the PVDF membrane with 1×PBST solution 3-5 times, 5min each time; the purpose is to wash off the milk and impurities adsorbed on the membrane. Then select the target primary antibody for dilution, and incubate the primary antibody and PVDF membrane together at room temperature for 2 hours or choose to incubate overnight on a shaker in a 4℃ refrigerator. After the incubation, wash the PVDF membrane with 1×PBST solution 3-5 times, 5min each time; the purpose of this step is to wash away the primary antibody that is not bound to the PVDF membrane. Finally, select the secondary antibody corresponding to the primary antibody and incubate it with the PVDF membrane at room temperature for 1 hour. Wash the PVDF membrane with 1×PBST solution 5-8 times, 5min each time. Then use ECL development imaging for analysis.

[0119] Conclusion

[0120] 1. SYVN1 is highly expressed in gastric cancer tissues, and its expression level is negatively correlated with survival time.

[0121] After SYVN1 immunohistochemical staining of multiple gastric cancer tissues and adjacent gastric cancer tissues, it was found that the expression level of SYVN1 in cancer tissues was significantly higher than that in adjacent gastric cancer tissues. In addition, the survival time of patients in the SYVN1 high expression group was significantly lower than that of patients in the low expression group. This suggests that SYVN1 is closely related to the progression and prognosis of gastric cancer and may become an important target for clinical anti-gastric cancer treatment.

[0122] 2. The small interfering RNA used in the present invention can specifically interfere with the target gene SYVN1.

[0123] The small interfering RNA sequence used in the present invention was transfected into gastric cancer cell lines HCG-27 and SGC7901, and the protein level of SYVN1 decreased to less than 50% of the control group 96 hours after transfection, indicating that the small interfering RNA sequence used in the present invention can knock down the target gene SYVN1.

[0124] 3. Interference with SYVN1 can significantly inhibit the proliferation, migration, invasion and cloning ability of gastric cancer cells.

[0125] Through CCK-8 experiments, the proliferation ability of gastric cancer cells was significantly inhibited after SYVN1 was knocked down using small interfering RNA; through cell scratch experiments, the migration ability of gastric cancer cells was significantly inhibited after SYVN1 was knocked down using small interfering RNA; through Transwell experiments, the invasion ability of gastric cancer cells was significantly inhibited after SYVN1 was knocked down using small interfering RNA; through cell soft agar cloning experiments, the cloning ability of gastric cancer cells was significantly inhibited after SYVN1 was knocked down using small interfering RNA.

[0126] In summary, the target SYVN1 targeted by the small interfering RNA involved in the present invention is highly expressed in gastric cancer, and downregulating the expression of SYVN1 by RNAi technology can significantly inhibit the proliferation, migration, invasion and cloning ability of gastric cancer cells. The technical method adopted by the present invention can provide a specific therapeutic drug for gastric cancer, which can help solve the problems of non-surgical treatment effect and poor prognosis of advanced gastric cancer, and has important application value.

[0127] Embodiment 2:

[0128] Application of inhibitory substances of E3 ubiquitin ligase SYVN1 in the preparation of drugs for treating gastric cancer.

[0129] A preferred embodiment is that the inhibitory substance is a small interfering RNA that downregulates the expression of the SYVN1 gene in gastric cancer cells.

[0130] A preferred embodiment is that the nucleotide sequence of the 5'-3' direction of the sense strand of the small interfering RNA is:

[0131] 5'-GCAUUGUCUCUCUUAUGUUZZ-3'Z=dT;

[0132] The nucleotide sequence of the antisense strand in the 5'-3' direction is:

[0133] 5'-AACAUAAGAGAGACAAUGCZZ-3', where Z=dT.

[0134] A recombinant vector is used in the preparation of a drug for treating gastric cancer, wherein the recombinant vector contains small interfering RNA.

[0135] The above description is only used to explain the preferred embodiments of the present invention, and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. Application of inhibitory substances of E3 ubiquitin ligase SYVN1 in the preparation of drugs for the treatment of gastric cancer.

2. Use of the inhibitor of E3 ubiquitin ligase SYVN1 according to claim 1 in the preparation of a drug for treating gastric cancer, characterized in that: The inhibitory substance is a small interfering RNA that downregulates the expression of the SYVN1 gene in gastric cancer cells.

3. Use of the inhibitor of E3 ubiquitin ligase SYVN1 according to claim 2 in the preparation of a drug for treating gastric cancer, characterized in that: The nucleotide sequence of the 5'-3' direction of the sense strand of the small interfering RNA is: 5'-GCAUUGUCUCUCUUAUGUUZZ-3'Z=dT; The nucleotide sequence of the antisense strand in the 5'-3' direction is: 5'-AACAUAAGAGAGACAAUGCZZ-3', where Z=dT.

4. Use of a recombinant vector in the preparation of a drug for treating gastric cancer, characterized in that: The recombinant vector comprises the small interfering RNA according to claim 2 or 3.

Images