Use of an inhibitory substance of e3 ubiquitin ligase syvn1 in the preparation of a gastric cancer treatment drug

Abstract

The application of the inhibiting substance of E3 ubiquitin ligase SYVN1 in the preparation of a stomach cancer treatment drug, the small interfering RNA used in the application is aimed at the target point SYVN1, the expression level of the target point SYVN1 in the cancer tissue of a stomach cancer patient is higher than that in the cancer-adjacent tissue, and the target point SYVN1 is negatively correlated with the survival time, and has unique clinical application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and relates to application of an E3 ubiquitin ligase SYVN1 inhibitor in preparation of a gastric cancer treatment drug. BACKGROUND

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

[0003] The current treatment methods for gastric cancer include surgical treatment, chemotherapy, radiotherapy, endoscopic treatment, etc., but each treatment method still has some limitations. In particular, drug treatment, although a large number of drugs for treating gastric cancer have been applied in clinical practice, has great side effects, is prone to drug resistance, and individual differences lead to limited efficacy. Therefore, it is of great significance to find a drug target for targeted gastric cancer treatment.

[0004] SYVN1 (HMG-CoA reductase degradation protein 1) located on the endoplasmic reticulum membrane, also known as HRD1, is a E3 ubiquitin ligase that can participate in the endoplasmic reticulum associated degradation (ERAD) pathway. 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 shown that SYVN1 plays a very important role in various malignant tumors.

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

[0006] The application provides an application of an inhibitor of E3 ubiquitin ligase SYVN1 in preparation of a gastric cancer treatment drug.

[0007] To achieve the above object and other related objects, the application provides a technical scheme that an application of an inhibitor of E3 ubiquitin ligase SYVN1 in preparation of a gastric cancer treatment drug.

[0008] Preferably, the inhibitor is small interfering RNA for down-regulating SYVN1 gene expression in gastric cancer cells.

[0009] Preferably, the nucleotide sequence of the sense strand of the small interfering RNA in the 5'-3' direction 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', wherein Z=dT in the sequence.

[0013] To achieve the above object and other related objects, the application provides a technical scheme that an application of a recombinant vector in preparation of a gastric cancer treatment drug, wherein the recombinant vector contains small interfering RNA.

[0014] Thanks to the above technical scheme, the application has the following advantages compared with the prior art:

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

[0016] 2. The application provides a new gastric cancer treatment method, and provides an effective treatment method for patients with advanced gastric cancer, and solves the technical problems of poor effect and poor prognosis of the existing treatment method for advanced gastric cancer.

[0017] 3. The small interfering RNA in the application can inhibit the proliferation, migration, invasion and clone formation ability of gastric cancer cells, and inhibit the occurrence and development of gastric cancer.

[0018] 3、The small interfering RNA adopted in the application is aimed at the target point SYVN1, the expression level of which in the cancer tissue of a gastric cancer patient is higher than that in the paracancer tissue, and is negatively correlated with the survival time, and has unique clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 : Figure A-B is the expression of SYVN1 in normal gastric tissue and gastric cancer tissue detected by immunohistochemical experiment, and the results show that the expression level of SYVN1 in the gastric cancer tissue is obviously higher than that in the normal tissue; Figure C-D is the expression of SYVN1 in the gastric cancer tissue and the paracancer tissue detected by immunohistochemical experiment, and the results show that the expression level of SYVN1 in the gastric cancer tissue is obviously higher than that in the paracancer tissue; Figure E is the expression of SYVN1 in the gastric cancer tissue and the paracancer tissue detected by immunoblotting, and the primary antibody in the figure is the SYVN1 antibody and the GAPDH antibody, and the results also show that the expression level of SYVN1 in the gastric cancer tissue is obviously higher than that in the paracancer tissue.

[0020] Figure 2 : The correlation between the expression of SYVN1 gene and the overall survival time is analyzed by using the survival time sample in the TCGA database. The results show that with the increase of the expression of SYVN1, the overall survival time of the cancer patient is significantly shortened.

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

[0022] Figure 4Figures A and B show the results of the CCK-8 assay, which showed that overexpression of SYVN1 promoted the proliferation of MGC803 and AGS cells, while knockdown of SYVN1 inhibited the proliferation of HCG-27 and SGC7901 cells. Figures C and D show the results of the soft agar cloning assay, which showed that overexpression of SYVN1 promoted the colony formation of MGC803 and AGS cells, while knockdown of SYVN1 inhibited the colony formation of HCG-27 and SGC7901 cells.

[0023] Figure 5 Figures A and B show the scratch assay results: overexpression of SYVN1 promotes the migration ability of MGC803 and AGS cells, while knockdown of SYVN1 inhibits the migration ability of HCG-27 and SGC7901 cells. Figures C and D show the Transwell assay results: overexpression of SYVN1 promotes the invasion ability of MGC803 and AGS cells, while knockdown of SYVN1 inhibits the invasion ability of HCG-27 and SGC7901 cells. Detailed Implementation

[0024] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in these embodiments.

[0025] Please see Figures 1-5 It should be noted that the structures, proportions, sizes, etc., illustrated in the accompanying drawings are merely for illustrative purposes to aid those skilled in the art and to provide a clear understanding of the invention. They are not intended to limit the scope of the invention and therefore have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to the size are not permitted. The following embodiments are provided to better understand the invention, but are not intended to limit it. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0026] The application of an inhibitor of the E3 ubiquitin ligase SYVN1 in the preparation of a gastric cancer therapeutic drug: The inhibitor of the E3 ubiquitin ligase SYVN1 is a small interfering RNA that downregulates the SYVN1 gene in gastric cancer cells. The gastric cancer therapeutic drug of this invention contains an inhibitor of SYVN1, which can solve the problems of poor efficacy and poor prognosis of traditional gastric cancer treatments.

[0027] In gastric cancer patients, the expression level of SYVN1 in cancerous 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 colony formation of gastric cancer cells.

[0028] The application of the reagent for inhibiting or down-regulating the expression of 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 E3 ubiquitin ligase SYVN1 gene is a small interfering RNA (siRNA) targeting E3 ubiquitin ligase SYVN1 gene, which can continuously inhibit or down-regulate the expression of the target gene in cells. The nucleotide sequence of the sense strand of the small interfering RNA is shown in SEQ ID NO. 1; the nucleotide sequence of the antisense strand is shown in SEQ ID NO. 2.

[0030] Sense strand:

[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 = dT in the sequence

[0036] The drug for treating gastric cancer can inhibit the proliferation, migration, invasion and clonogenicity 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 for SYVN1 gene used was synthesized by Shanghai Shengong Bioengineering Co., Ltd., and was confirmed to be an effective sequence capable of down-regulating the expression of SYVN1 in gastric cancer cells.

[0038] The SYVN1 small interfering RNA oligonucleotide sequence is a freeze-dried powder, which is stored at -20℃ and dissolved with DEPC water before use.

[0039] The principle of the present application is that the small interfering RNA is transfected into cells using a transfection reagent, and under the action of a cellular RNA helicase, the small interfering RNA is unzipped into a sense strand and an antisense strand; the antisense strand is recruited and integrated into a RNA-induced silencing complex (RISC) by the RISC; the antisense strand of the small interfering RNA is combined with a target mRNA by the principle of base complementarity to form double stranded RNA (dsRNA), and this combination recruits an endonuclease to cut the target mRNA into fragments, thereby preventing it from being translated into a protein, resulting in down-regulation of gene expression.

[0040] The application of a recombinant vector in the preparation of a gastric cancer treatment drug, wherein the recombinant vector comprises the small interfering RNA targeting the E3 ubiquitin ligase SYVN1 gene.

[0041] A gastric cancer treatment drug, wherein the gastric cancer treatment drug comprises the small interfering RNA targeting the E3 ubiquitin ligase SYVN1 gene.

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

[0043] Experimental materials involved in the experiment:

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

[0045] 2. Experimental materials, reagents and kits

[0046] DMEM medium: Wuhan Sevier Biological Technology Co., Ltd.; fetal bovine serum: Thermo Fisher Scientific; RIPA lysis buffer: Shanghai Biyun Tian Biological Technology Co., Ltd.; PMSF: Shanghai Biyun Tian Biological Technology Co., Ltd.; protease inhibitor Cocktail: Shanghai Biyun Tian Biological Technology Co., Ltd.; ECL developing solution: Wuhan Sevier Biological Technology Co., Ltd.; SDS-PAGE gel preparation kit: Shanghai Biyun Tian Biological Technology Co., Ltd.; PVDF membrane: Millipore; transfection reagent: Thermo Fisher Scientific; anhydrous ethanol: National Pharmaceutical Group Chemical Reagent Co., Ltd.; Transwell chamber: Corning; 1x crystal violet staining solution: Shanghai Biyun Tian Biological Technology Co., Ltd.; ready-to-use immunohistochemical detection kit: Beijing Zhongshanjingqiao Biological Technology 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 biosafety cabinet: Boke Group Co., Ltd., China; BD series CO2 cell culture incubator: Binder GmbH, Germany; 224s precision electronic balance: Sartorius GmbH, Germany; electronic analytical balance: Desert Eagle Co., Ltd., China; -80℃ ultra-low temperature medical freezer: Haier Corporation, China; 4℃ freezer: Haier Corporation, China; ordinary room temperature shaker: Taicang Hualida Experimental Equipment Co., Ltd.; 4℃ standard product display cabinet: Beijing Fuyi Co., Ltd.; EVOS type inverted fluorescence microscope purchased from Thermo Fisher Scientific, USA; VE180 and EPS300 type vertical electrophoresis tanks and electrophoresis apparatus purchased from Shanghai Tianneng Co., Ltd.; G3E microwave oven purchased from Midea Group, China; 0-150MM high-precision vernier calipers purchased from Shanghai Minate Instrument Co., Ltd. Detailed implementation method:

[0051] 1. Immunohistochemical analysis showed that the expression level of SYVN1 in gastric cancer tissue was significantly higher than that in adjacent normal tissue. Figure 1 C and Figure 1 As shown in D).

[0052] (1) Fixation, dehydration, embedding, sectioning (4μm), slide retrieval (46℃-48℃), and baking (90℃, 20min).

[0053] (2) Dewaxing: Immerse the slides in xylene I / II and place them in a 65°C oven for 1 hour, then return them to a fume hood to cool slowly. Then, pass them through different concentration gradients of alcohol, such as 100%, 90%, 80%, and 70%, for 5 minutes each time. After that, wash them in double-distilled deionized water for 5 minutes. Finally, wash them with PBS solution about 3 times, with an interval of 5 minutes each time.

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

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

[0056] (5) Wash the slides with PBS buffer about 3 times, with an interval of 5 minutes each time. At the same time, prepare 0.5% periodic acid in the dark: that is, add 0.1g of periodic acid to 20mL of double-distilled deionized water.

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

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

[0059] (8) Prepare the primary antibody during the blocking period 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 with your hand. Carefully add the primary antibody, place the slide in a humidified chamber, and incubate overnight at 4°C.

[0061] (10) Take out the humidified box the next day. If there are still a lot of antibodies on the slide, warm it at 37°C for 30 minutes. If there are fewer antibodies, warm it at room temperature for 30 minutes.

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

[0063] (12) Add secondary antibody using a three-step method: first add yellow IgG and incubate at 37°C for about 30 minutes, then add red horseradish enzyme and incubate again at 37°C for 30 minutes, and finally wash with PBST for 30 minutes 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 the microscope. Start timing as you add the DAB and record the time it takes for the slide to turn brown under the microscope. Rinse gently under running water for a few moments, then stain with hematoxylin for 40 seconds. Finally, rinse the slide under running water until the water is colorless. Observe the slide under the microscope. If the cell nuclei are too dark, continue rinsing with running water; otherwise, if the cell nuclei are too light, repeat the staining.

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

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

[0068] (17) Finally, seal the film with neutral resin, being careful not to create air bubbles.

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

[0070] Gastric cancer tissue samples with complete survival times were downloaded from the TCGA database. They were divided into high-SYVN1 expression and low-SYVN1 expression groups based on the median SYVN1 expression level, and Kaplan-Meier survival curve analysis was performed. Figure 2 Kaplan-Meier survival curves for gastric cancer patients are presented. As shown in the figure, SYVN1 gene expression is correlated with overall survival; as SYVN1 expression increases, the overall survival of gastric cancer patients significantly decreases (P < 0.01). Later, as the number of patients followed up gradually decreased, the survival rates of gastric cancer patients in the high and low SYVN1 expression groups tended to be similar.

[0071] 3. Overexpression of SYVN1 promotes the proliferation of gastric cancer cells, while knockdown of 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) Plant 6-hole or 24-hole boards the night before.

[0074] (2) Transfection was performed on the second day, while for cells transfected with exogenous plasmids, the seed plate was digested 24-48 hours later.

[0075] (3) After the cells were digested with trypsin, they were resuspended in 1 mL of culture medium and counted three times. The average value was then used for calculation. Approximately 3000-5000 cells were seeded into each well, and five 96-well plates were seeded at five time points. The plates were then placed in a cell culture incubator for further culture.

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

[0077] (5) Gastric cancer cell lines generally adhere to the plate in about 4-6 hours. Remove a 96-well plate from the incubator and discard the old culture medium. Perform the incubation in the dark, adding 100 μL of the prepared culture medium to each well, with three replicates per group. Additionally, three background wells should be prepared, containing culture medium without CCK-8 reagent, to minimize error. Wrap the lid with aluminum foil, then close the lid and continue incubating 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 in each well. Record this value as the 0h value. Subsequently, at four time points of 24h, 48h, 72h and 96h, add culture medium containing CCK-8 reagent and perform the same operation as above.

[0079] (7) Finally, a relative proliferation curve is plotted 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 knockdown of 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 assay is to evaluate the migration ability of normal gastric cancer cell lines and gastric cancer cell lines that knock out genes and overexpress SYVN1. First, prepare the ruler and pen needed for the experiment and sterilize them in a sterile laminar flow hood under UV light for 30 minutes. All other instruments need to be sterilized at high temperature. Then, using a pen and a ruler, draw a horizontal line across the back of each well of a 6-well plate at a suitable distance. Each well should have at least five parallel lines. Seed the cells into the wells of a new sterile 6-well plate. When the cell density is between 80% and 90%, remove the old culture medium from the wells using a pipette. Then, using a 20 μL pipette with a yellow tip, carefully draw scratches perpendicular to the five horizontal lines on the back of the plate, about 3-4 scratches per well, referring to the ruler. Gently wash the 6-well plate twice with PBS solution to remove dead and detached cells. Add culture medium containing 5% serum and a small amount of antibiotics to the 6-well plate and incubate at 37°C with 5% CO2. The six-well plates were removed 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 knockdown of SYVN1 inhibits the invasive ability of gastric cancer cells. Figure 5 C and Figure 5 As shown in D).

[0084] The Transwell experimental method is as follows:

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

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

[0087] (3) Cell inoculation: First, place 500 μL of culture medium containing 20% ​​serum (with appropriate amount of antibiotics) in the lower well of each chamber. Then, count the cells and inoculate each chamber with approximately 100 μL of culture medium containing 4 × 10⁶ cells / mL. 4 The cell suspension contains individual cells. Carefully place the chambers into the wells, ensuring there are no air bubbles between the chambers and the culture medium in the wells. If air bubbles are found during the procedure, carefully lift the chamber, remove the air bubbles, and then gently place the chamber back into the culture well.

[0088] (4) After incubating in an incubator for 48 hours, fix the chambers with formaldehyde at room temperature for 30 minutes, then stain with crystal violet dye at room temperature for 30-60 minutes. Rinse with running water, and then gently wipe away any excess matrix gel and crystal violet dye from the bottom of the chambers with a cotton swab. Finally, invert the chambers and observe and photograph them under a microscope.

[0089] 6. Overexpression of SYVN1 promotes the clonal formation ability of gastric cancer cells, while knockdown of SYVN1 inhibits the clonal formation ability of gastric cancer cells. Figure 4 C and Figure 4 As shown in D).

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

[0091] The purpose of the soft agar cell clonal assay is to determine the malignant colony-forming ability of cells. Control cells, gastric cancer cell lines overexpressing a certain gene, and gene knockout-treated cells were resuspended and seeded at a density of 400 cells per well in new six-well plates lined with agarose gel. The plates were then incubated in a sterile incubator at 37°C and 5% CO2. During incubation, the agarose gel at the bottom of the wells was frequently observed for turbidity or contamination. After approximately 7-11 days, multiple visible clonal cell clusters would appear at the bottom of the six-well plates, at which point the culture could be terminated. The six-well plates were then gently washed 2-3 times with 1×PBS to remove impurities. The cell clusters were then fixed with 4% paraformaldehyde at room temperature for 30-60 minutes. 1-2 mL of crystal violet solution was added to the six-well plates and stained at room temperature for 30 minutes. After staining, the six-well plates were gently rinsed with water to remove the crystal violet, and finally dried and photographed upside down.

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

[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) The SYVN1 small interference sequence was purchased from Shanghai Sangon Biotech Co., Ltd.

[0097] 2. Experimental materials, reagents and kits

[0098] DMEM medium: Wuhan Sewell Biotechnology Co., Ltd.; Fetal bovine serum: Thermo Fisher Scientific Co., Ltd.; Transfection reagent: Shanghai Sangon Biotech Co., Ltd.

[0099] 3. Main instruments and equipment

[0100] A2 biosafety cabinet: Boke Group Co., Ltd., China; BD series carbon dioxide cell culture incubator: Binder GmbH, Germany; -80℃ ultra-low temperature medical freezer: Haier Corporation, China; 4℃ freezer: Haier Corporation, China; ordinary room temperature shaker: Taicang Hualida Experimental Equipment Co., Ltd.; 4℃ standard product display cabinet: Beijing Fuyi Co., Ltd.; EVOS type inverted fluorescence microscope purchased from Thermo Fisher Scientific, USA; VE180 and EPS300 type vertical electrophoresis tanks and electrophoresis apparatus purchased from Shanghai Tianneng Co., Ltd.; centrifuge purchased from Hunan Xiangyi Centrifuge Instrument Co., Ltd. Detailed implementation method:

[0102] 1. Small interfering RNA transfection

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

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

[0105] (3) Transfection procedure: Taking a 60mm culture dish as an example. Prepare two centrifuge tubes, each containing 500μL of serum-free DMEN medium. Add 10μL of transfection reagent to one tube, gently mix by pipetting, and incubate at room temperature for 5 min; add 10μL of small interfering RNA working solution to the other tube. Mix the two liquids, pipette to mix, and incubate at room temperature for 20 min. Then, discard the medium in the culture dish to be transfected, add 1mL of the above mixture and 3mL of antibiotic-free medium containing 10% fetal bovine serum, and transfer to a cell culture incubator to stand for 24 h.

[0106] (4) Cell treatment after transfection: 24 hours after cell transfection, the culture medium and transfection reagent were discarded, the cells were digested and counted. The cells were then re-seeded into plates for subsequent experiments according to the target.

[0107] 2. Verification of SYVN1 knockdown efficiency in gastric cancer cells: The knockdown efficiency of small interfering RNA was verified using Western blotting. The Western blotting method is as follows:

[0108] (1) Protein sample preparation

[0109] After 24 hours of cell transfection or other necessary experimental procedures, gently aspirate and discard the old culture medium from the culture dish using a pipette. Carefully wash the cells twice with sterile 1×PBS buffer to remove dead cells and impurities. Then, add 1-2 mL of cell lysis buffer containing protease inhibitors to the cell culture dish and place it on ice for 20-30 minutes to ensure thorough cell lysis. Mix the lysis buffer thoroughly and collect it into 1.5 mL EP tubes using a pipette. Simultaneously, prepare boiling water at 100°C and immerse the tubes for 15 minutes to ensure complete protein denaturation.

[0110] (2) SDS-PAGE gel preparation

[0111] SDS-PAGE gel preparation

[0112]

[0113] (3) Electrophoresis

[0114] Choose the size of the pre-stained protein marker based on the molecular weight of the target protein; after loading the sample, cap the gel and turn on the power. Initially use 80V-90V, and once the pre-stained protein markers separate, switch to 120V-130V to continue electrophoresis. The time to turn off the electrophoresis apparatus depends on the time it takes for the loading buffer to reach the bottom of the SDS-PAGE gel, generally around two hours.

[0115] (4) Transfer membrane

[0116] After the electrophoresis steps described above, carefully cut the SDS-PAGE gel and transfer it to the prepared transfer buffer. Depending on the protein molecular weight, perform wet transfer at a low temperature in the transfer tank 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 a suitable container to avoid contamination. Weigh 1g of skim milk powder and dissolve it in a 50mL centrifuge tube containing 20mL of 1×PBST. Incubate the milk and PVDF membrane at room temperature for 1 hour. After incubation, wash the PVDF membrane 3-5 times with 1×PBST solution, 5 minutes each time; this is to remove adsorbed milk and impurities from the membrane. Then, dilute the target primary antibody and incubate it together with the PVDF membrane at room temperature for 2 hours or overnight on a shaker at 4°C. After incubation, wash the PVDF membrane 3-5 times with 1×PBST solution, 5 minutes each time; this step is to remove any primary antibody that has 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 5-8 times with 1×PBST solution, 5 minutes each time. Then, use ECL imaging for analysis.

[0119] III. Conclusion

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

[0121] Immunohistochemical staining of SYVN1 in multiple gastric cancer tissues and adjacent normal tissues revealed that SYVN1 expression levels were significantly higher in cancerous tissues than in adjacent normal tissues. Furthermore, patients in the high SYVN1 expression group had significantly shorter survival times than those 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 therapy.

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

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

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

[0125] CCK-8 assays showed that knocking down SYVN1 with small interfering RNA significantly inhibited the proliferation of gastric cancer cells; cell scratch assays showed that knocking down SYVN1 with small interfering RNA significantly inhibited the migration of gastric cancer cells; Transwell assays showed that knocking down SYVN1 with small interfering RNA significantly inhibited the invasion of gastric cancer cells; and soft agar cloning assays showed that knocking down SYVN1 with small interfering RNA significantly inhibited the colony-forming ability of gastric cancer cells.

[0126] In summary, the small interfering RNA involved in this invention targets SYVN1, which is highly expressed in gastric cancer. Downregulating SYVN1 expression through RNAi technology can significantly inhibit the proliferation, migration, invasion, and colony formation of gastric cancer cells. The technology employed in this invention can provide a specific therapeutic agent for gastric cancer, helping to address the problems of poor non-surgical treatment outcomes and prognosis in advanced gastric cancer, and has significant application value.

[0127] Example 2:

[0128] Application of inhibitors 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 as follows: the nucleotide sequence of the positive strand of the small interfering RNA in the 5'-3' direction is:

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

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

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

[0134] The use of a recombinant vector in the preparation of a gastric cancer therapeutic drug, wherein the recombinant vector contains small interfering RNA.

[0135] The above description is merely a preferred embodiment for explaining the present invention and is not intended to limit the present invention in any way. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included within the scope of protection intended by the present invention.

Claims

1. Application of inhibitors of E3 ubiquitin ligase SYVN1 in the preparation of drugs for treating gastric cancer; The inhibitory substance is a small interfering RNA that downregulates the expression of the SYVN1 gene in gastric cancer cells; The nucleotide sequence of the small interfering RNA in the 5'-3' direction of the positive strand is as follows: 5'-GCAUUGUCUCUCUUAUGUUZZ-3' Z= dT; The nucleotide sequence in the 5'-3' direction of the antisense strand is as follows: 5'-AACAUAAGAGAGACAAUGCZZ-3', where Z = dT in the sequence.

2. The application of a recombinant vector in the preparation of a gastric cancer therapeutic drug, characterized in that: The recombinant vector contains the small interfering RNA as described in claim 1.

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