Research method of influence mechanism of TMEM91 gene on renal clear cell carcinoma

Abstract

The invention discloses a method for researching an influence mechanism of a TMEM91 gene on renal clear cell carcinoma, and relates to the technical field of biomedicine. Comprising the following steps: S1, preparing materials; s11, cell line selection: selecting a human embryonic kidney cell line HEK 293T, a human kidney proximal tubular epithelial cell HK2, and human kidney cancer cell lines 786-O, A498, ACHN and OSRC-2; s12, a culture medium and a reagent, wherein an RPMI 1640 culture medium is used for 786-O; other cell lines use a DMEM culture medium; 10% of fetal calf serum and 50 mg / ml of penicillin / streptomycin are added into all the culture media. By revealing up-regulated expression of the TMEM91 gene in the renal clear cell carcinoma and the influence of the TMEM91 gene on proliferation, migration and apoptosis of cancer cells, a potential biomarker and a treatment target are provided, and improvement of early diagnosis and treatment effects of ccRCC is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method for studying the influence mechanism of TMEM91 gene on renal clear cell carcinoma. Background Art

[0002] Renal cell carcinoma (RCC) is a common malignant tumor in the urinary system, among which clear cell renal carcinoma (ccRCC) is the subtype of the vast majority of RCC, accounting for about 75% of all RCC cases. The treatment of early renal cancer is mainly partial nephrectomy or radical nephrectomy, with a five-year overall survival rate of up to 96%. However, about one-third of ccRCC patients diagnosed with localized disease have tumor recurrence or metastasis after surgical resection, and the prognosis is poor.

[0003] Although breakthroughs have been made in the molecular mechanisms and treatment strategies of ccRCC in recent years, the prognosis of ccRCC patients remains poor and the incidence continues to increase. Therefore, there is an urgent need to find effective ccRCC prognostic biomarkers to provide new basis for the early diagnosis and treatment of ccRCC. Summary of the invention

[0004] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a research method for the mechanism of the influence of TMEM91 gene on renal clear cell carcinoma.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for studying the mechanism of the effect of TMEM91 gene on renal clear cell carcinoma, comprising:

[0007] S1: Material preparation;

[0008] S11: Cell line selection, using human embryonic kidney cell line HEK 293T, human kidney proximal tubule epithelial cell HK2, and human renal cancer cell lines 786-O, A498, ACHN, OSRC-2;

[0009] S12: Culture medium and reagents, RPMI 1640 medium was used for 786-O; DMEM medium was used for other cell lines; all culture media were supplemented with 10% fetal bovine serum and 50 mg / ml penicillin / streptomycin;

[0010] S2: vector construction and gene transfection;

[0011] S21: Flag-TMEM91 plasmid construction;

[0012] S22: cell transfection experiment;

[0013] S3: Gene silencing and effect verification;

[0014] S31: siRNA design and transfection:

[0015] Design specific siRNA and negative control siRNA according to the TMEM91 gene sequence; use GP-transfect-mate transfection reagent to transfect siRNA into renal cancer cells in proportion;

[0016] S32: Knockdown efficiency verification: The knockdown efficiency of si-TMEM91 was detected by Western blotting WB, and the experiment was repeated to ensure the reproducibility and reliability of the results;

[0017] S4: Functional experimental analysis;

[0018] S41: Cell proliferation experiment, using MCE CCK-8 kit to evaluate cell proliferation ability; at different time points: 0h, 12h, 24h, 48h, measure absorbance and calculate cell proliferation rate;

[0019] S42: Cell migration experiment, using transwell chamber to perform cell migration experiment; after culturing under appropriate conditions, the migration ability was analyzed by crystal violet staining and counting the cells that migrated to the lower layer under a microscope;

[0020] S43: Cell apoptosis detection, apoptosis rate was analyzed by flow cytometry; cells were double stained with Annexin V / PI, and the proportion of apoptotic cells was analyzed by flow cytometry;

[0021] S5: protein expression level analysis;

[0022] S51: immunoblot analysis;

[0023] S52: Result analysis.

[0024] Preferably, in S1, the cell line is cultured in a cell incubator at 37°C containing 5% CO2, and the cell morphology and growth state are regularly observed to keep the cells in a good growth environment.

[0025] Preferably: In S21, the Flag-TMEM91 plasmid is constructed, comprising:

[0026] TMEM91 sequence information was downloaded from NCBI, and CDS and restriction sites were analyzed using SnapGene software;

[0027] Design specific primers and add homology arms, confirm the specificity through blast comparison, and then have BGI synthesize the primers;

[0028] Using HEK 293T cell cDNA as a template, TMEM91 CDS was amplified by PCR, connected to the CMV-Flag vector, and purified plasmid was obtained by plasmid extraction.

[0029] Preferably: in said S22, the cell transfection experiment comprises:

[0030] Flag-TMEM91 plasmid was transiently transfected into the target cell line using Lipofectamine2000;

[0031] A control group and an experimental group were set up to conduct preliminary tests on transfection efficiency and gene expression levels, respectively.

[0032] Preferably: the cell proliferation experiment is as follows:

[0033] S411: Cell seeding: cells in good condition were digested with trypsin and counted, and seeded in a 96-well plate at a density of 1000 cells per well; at least 3 replicates were set for each experimental condition to ensure the accuracy of the data;

[0034] S412: Cell attachment: Allow cells to grow in 96-well plates for 24 hours to ensure they are fully attached and proliferating before experimental treatment;

[0035] S413: Experimental treatment, adding different concentrations of drugs, gene silencing or overexpression treatment;

[0036] S414: Proliferation detection, using the MCE CCK-8 kit, CCK-8 solution was added to each well at a ratio of 1:100, and then incubated at 37°C for 1 hour; the absorbance value at 450nm was measured using an ELISA reader; measurements were performed at different time points after the start of the experiment: 0h, 12h, 24h, and 48h to evaluate cell proliferation.

[0037] Preferably: the cell migration experiment is as follows:

[0038] S421: Preparation of transwell chamber: Take a transwell chamber, the upper and lower chambers are separated by a polycarbonate membrane with a pore size of 8.0 μm; ensure that the chamber is sterile and clean;

[0039] S422: Cell preparation and planting: resuspend the corresponding treated cells in serum-free medium and adjust the cell density to the appropriate concentration; add the cell suspension to the upper chamber, and add the medium containing 10% FBS as a chemical attractant to the lower chamber;

[0040] S423: Cell culture, incubated at 37°C with 5% CO2 for 48 hours to allow sufficient time for cells to migrate through the polycarbonate membrane;

[0041] S424: Fixation and staining: remove the upper chamber, wash with PBS to remove the non-migrated cells, then fix the cells that migrated to the bottom of the upper chamber with 4% paraformaldehyde, and stain the cells with 1% crystal violet solution. The staining time is adjusted according to the cell type and the strength of the staining solution;

[0042] S425: Observation and analysis of results: Use a cotton swab to gently wipe off the non-migrated cells on the inner side of the upper chamber, then randomly select fields of view under a microscope to take pictures and count the migrated cells, and statistically analyze the cell migration ability.

[0043] Preferably, the cell apoptosis detection is as follows:

[0044] S431: Cell collection: collect the cell culture medium from the culture plate and digest the cells with EDTA-free trypsin; mix the digested cells with the previously collected culture medium and collect the cells by gentle centrifugation;

[0045] S432: Cell washing: wash the cells twice with cold PBS, discard the supernatant after centrifugation, and ensure that the cell pellet is intact;

[0046] S433: Staining and analysis: resuspend the cells with staining buffer, add Annexin V and PI dyes according to the kit instructions, and incubate at room temperature in the dark for 15 minutes; analyze using flow cytometry to record the proportion of early apoptotic and late apoptotic cells.

[0047] Preferably: the immunoblot analysis comprises:

[0048] After extracting total cell protein, the protein concentration was determined using a BCA kit; separation gel and concentration gel of corresponding concentrations were prepared according to the size of the target protein;

[0049] After electrophoresis and membrane transfer, incubation was performed with specific antibodies, color was developed with developer, and the results were recorded using a gel imager.

[0050] Preferably: the result analysis includes:

[0051] The expression level changes of various proteins including TMEM91 under gene overexpression or silencing conditions were analyzed to analyze their biological functions in ccRCC.

[0052] The beneficial effects of the present invention are:

[0053] 1. The present invention provides a potential biomarker and therapeutic target by revealing the upregulated expression of TMEM91 gene in renal clear cell carcinoma and its effect on cancer cell proliferation, migration and apoptosis, which helps to improve the early diagnosis and treatment of ccRCC.

[0054] 2. The present invention not only discovered the upregulated expression of TMEM91 through bioinformatics analysis, but also systematically verified the specific role of TMEM91 in the proliferation, migration and apoptosis of renal cancer cells through a series of in vitro experiments, ensuring the reliability and scientificity of the research results. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is a graph showing the difference in expression of TMEM91 between ccRCC cell lines and normal renal tubular epithelial cell lines detected by WB in the present invention;

[0056] Figure 2 This is a WB diagram verifying the efficiency of knocking down TMEM91 in 786-O cells in the present invention;

[0057] Figure 3 This is a schematic diagram of the CCK8 experiment for detecting cell proliferation ability in the present invention;

[0058] Figure 4 Schematic diagram of detecting apoptosis of 786-O cells by flow cytometry after knocking down TMEM91 in the present invention;

[0059] Figure 5 Schematic diagram of detecting apoptosis of 786-O cells by flow cytometry after overexpression of TMEM91 in the present invention;

[0060] Figure 6 This is a schematic diagram of the changes of apoptosis-related proteins in 786-O cells detected by WB in the present invention;

[0061] Figure 7 This is a schematic diagram of detecting the migration ability of cells in the cell migration experiment of the present invention;

[0062] Figure 8 This is a schematic diagram of the changes in EMT-related marker proteins in 786-O cells detected by WB after knocking down TMEM91 expression in the present invention;

[0063] Fig. 9 This is a schematic diagram of the changes in EMT-related marker proteins detected by WB in 786-O cells after overexpression of TMEM91 in the present invention. DETAILED DESCRIPTION

[0064] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0065] Embodiment 1:

[0066] A method for studying the mechanism of the effect of TMEM91 gene on renal clear cell carcinoma, specifically comprising:

[0067] 1. Material preparation:

[0068] 1. Cells

[0069] Human embryonic kidney cell line HEK 293T, human kidney proximal tubule epithelial cell HK2, human renal cancer cell lines 786-O, A498, ACHN, OSRC-2 (from ATCC cell bank, purchased by the research team and stored in liquid nitrogen for a long time). Except for 786-O which used RPMI 1640 medium (Gibco, USA), the rest of the cells used DMEM medium (Gibco, USA), and then added 10% fetal bovine serum (biochanel, China) and 50mg / ml penicillin / streptomycin (HyClone, USA) to the medium, and the cells were cultured in a cell incubator at 37°C with a 5% CO2 concentration;

[0070] 2. Reagents

[0071] Restriction enzymes EcoRⅠ, XhoⅠ (NEB, USA), KOD-plus-neo polymerase (TOYOBO, Japan), DNA purification kit (Omega, China), plasmid extraction kit (Kangwei Century, China), Lipofectamine2000 transfection reagent (Invitrogen, USA), GP-transfect-mate (Jima, China). Experimental related antibodies are as follows: rabbit TMEM91 (abcam, UK), mouse β-actin (Quanshijin, China), mouse Flag antibody (ABclonal, China), rabbit Bcl-2 (abmart, China), rabbit Bax (abmart, China), rabbit caspase-3 (abmart, China), rabbit caspase-9 (abmart, China), rabbit cleaved-caspase-3 (abmart, China), rabbit E-cadherin (proteintech, China), rabbit N-cadherin, rabbit Vimentin (bioworld, China);

[0072] 3. Instruments

[0073] NanoDrop spectrophotometer (Thermo Fisher, USA), microplate reader (ThermoFisher, USA), protein electrophoresis and transfer tank (Bio-Rad, USA), gel imager ChemiDocTM XRS+ (Bio-Rad, USA), DMi8 inverted fluorescence microscope (Leica, Germany).

[0074] 2. Experimental Methods

[0075] 1. Flag-TMEM91 plasmid construction and transfection

[0076] The longest human variant of TMEM91 (NM_001098821.2) was screened from the NCBI Information Center, and the sequence information was downloaded and opened with SnapGene software to analyze the CDS and restriction sites. Primers were then designed and appropriate homology arms were added. After blast comparison, primers were synthesized by BGI, and TMEM91 was obtained by PCR reaction and DNA purification using HEK 293T cell cDNA as a template. The target fragment TMEM91 was ligated to the CMV-Flag vector digested with EcoRⅠ and XhoⅠ, and Flag-TMEM91 was obtained after plasmid extraction. Lipofectamine2000 was used to transiently transfect cells with Flag-TMEM91.

[0077] The primer sequences are as follows:

[0078] TMEM91 CDS F:5'-CTGTGATCGTGAGCGGAAAC-3'

[0079] TMEM91 CDS R:5'-TTGGCCCAAGCCTTGTTG-3'

[0080] 2. si-TMEM91 transfection

[0081] si-TMEM91 and negative control were synthesized by Shanghai Jima. The siRNA dry powder was diluted with DEPC water to 20uM and frozen in aliquots. The final concentration was 100nM in a 6-well plate and transfected with GP-transfect-mate transfection reagent at an appropriate ratio.

[0082] The siRNA sequences are as follows

[0083] si-NC: 5'-UUCUCCGAACGUGUCACGUTT-3'

[0084] si-TMEM91#1:5'-GAUGUUGAGGACAUGUCAUCC-3'

[0085] si-TMEM91#2:5'-CCCUAGUCUUCGUGAGCUU-3'

[0086] 3. Immunoblotting

[0087] The cell proteins were extracted with homemade SDS lysis buffer (1% SDS, 50mM Tris-HCl, 150mM NaCl, 1mM EDTA, 2mM Vanadate, 1mM PMSF, Inhibitor Cocktail), and the protein concentration was determined by BCA kit (ThermoFisher, USA). 15% separation gel and 4% concentration gel were prepared, and the loading system was configured according to the determined protein concentration. After transfer with PVDF membrane (Millipore, USA), the membrane was blocked with 5% skim milk powder (CST, USA) at 4°C for 4 hours, and the primary antibody was blocked at 4°C overnight. The membrane was washed with TBST for 3 times and incubated with the corresponding species secondary antibody at room temperature for 1 hour. After the end, it was washed with TBST for 3 times and the membrane was developed and exposed in a gel imager with developer (Tian Di Renhe, China).

[0088] 4. Cell proliferation

[0089] After digesting the cells to be tested, count 1000 cells and place them in a 96-well plate. After the cells adhere to the wall, perform the experiment according to the instructions of the MCE CCK-8 kit. Add the reagent at a ratio of 1:100 at 0h, 12h, 24h, and 48h, incubate at 37℃ for 1h, and then detect the absorbance at 450nm with an enzyme reader.

[0090] 5. Cell Migration

[0091] Separate the upper and lower chambers of the transwell, count the same cells, make a cell suspension with serum-free medium and add it to the upper chamber, and add medium containing 10% serum to the lower chamber. Incubate in a 37°C incubator for 48 hours. After the incubation, wash the upper chamber with PBS, fix it with 4% paraformaldehyde, and then stain it with 1% crystal violet. After staining, gently wipe the unmigrated cells in the upper chamber with a cotton swab, observe the cell number and morphology with a conventional microscope, and calculate the cell migration rate.

[0092] 6. Apoptosis

[0093] After the cells to be tested in the six-well plate are fully grown, the cell culture medium is collected, the cells are digested with EDTA-free trypsin, and then the cells are blown off with the collected culture medium, centrifuged at 1000rpm for 5min, the supernatant is discarded, the cell pellet is resuspended with 1ml PBS, and centrifuged again at 1000rpm for 5min. This is repeated twice, and the cell pellet is resuspended with 500ul PBS for the last time and sent to the flow cytometry platform to detect cell apoptosis.

[0094] 8. Statistical analysis

[0095] All experiments were repeated three times. GraphPad Prism 8 was used to draw the graphs. Unpaired Student's T analysis was performed. The data dispersion was expressed as mean ± standard deviation (SD). The test level was α = 0.05. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 indicated statistically significant differences.

[0096] 3. Results

[0097] like Figure 1 As shown, WB detected the difference in the expression of TMEM91 between ccRCC cell lines and normal renal tubular epithelial cell lines; (*P<0.05, **P<0.01, ***P<0.001);

[0098] The results showed that compared with normal renal tubular epithelial cells HK2, TMEM91 protein levels were highly expressed in four ccRCC cell lines (786O, A498, ACHN, OSRC-2), and the differences were statistically significant (P<0.05).

[0099] like Figure 2 As shown, WB verified the efficiency of knocking down TMEM91 in 786-O cells, (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0100] The experiment was repeated three times and all showed that the siRNA targeting TMEM91 had a knockdown efficiency at the protein level, and the difference was statistically significant.

[0101] like Figure 3 As shown, the expression of TMEM91 was knocked down in 786-O cells, and the cell proliferation ability was detected by CCK8 experiment (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0102] Compared with the control group, the proliferation ability of renal cancer cells with TMEM91 knockdown was weakened.

[0103] like Figure 4 As shown, TMEM91 was knocked down, and apoptosis of 786-O cells was detected by flow cytometry (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0104] The apoptosis rate of renal cancer cells after TMEM91 knockdown was significantly higher than that in the control group.

[0105] like Figure 5As shown, TMEM91 was overexpressed, and apoptosis of 786-O cells was detected by flow cytometry (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0106] Overexpression of TMEM91 reduced the apoptosis rate of renal cancer cells.

[0107] like Figure 6 As shown, after knocking down TMEM91 expression, WB detected the changes of apoptosis-related proteins in 786-O cells;

[0108] After knocking down TMEM91 expression, the expression levels of Caspase3, Cleaved-Caspase3, Caspase9, and Bax pro-apoptotic proteins were significantly increased compared with the control group, further verifying that knocking down TMEM91 promoted renal cancer cell apoptosis.

[0109] like Figure 7 As shown, the expression of TMEM91 was knocked down in 786-O cells, and the cell migration ability was detected by cell migration experiment (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0110] Knockdown of TMEM91 expression significantly weakened the migration ability of renal cancer cells.

[0111] like Figure 8 As shown, after knocking down TMEM91 expression, WB detected the changes of EMT-related marker proteins in 786-O cells;

[0112] After knocking down TMEM91, the expression of E-cadherin increased, while the expression of N-cadherin and Vimentin decreased, that is, knocking down TMEM91 can reduce the invasive ability of renal cancer cells.

[0113] like Fig. 9 As shown, after overexpression of TMEM91, WB was used to detect the changes of EMT-related marker proteins in 786-O cells;

[0114] After overexpression of TMEM91, the expression of E-cadherin decreased, while the expressions of N-cadherin and Vimentin increased, indicating that overexpression of TMEM91 can enhance the invasive ability of renal cancer cells.

[0115] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for studying the mechanism of TMEM91 gene's effect on renal clear cell carcinoma. comprising, characterized in that, include: S1: Material preparation; S11: Cell line selection, using human embryonic kidney cell line HEK 293T, human kidney proximal tubule epithelial cell HK2, and human renal cancer cell lines 786-O, A498, ACHN, OSRC-2; S12: Culture medium and reagents, RPMI 1640 medium was used for 786-O; DMEM medium was used for other cell lines; all culture media were supplemented with 10% fetal bovine serum and 50 mg / ml penicillin / streptomycin; S2: vector construction and gene transfection; S21: Flag-TMEM91 plasmid construction; S22: cell transfection experiment; S3: Gene silencing and effect verification; S31: siRNA design and transfection: Design specific siRNA and negative control siRNA according to the TMEM91 gene sequence; use GP-transfect-mate transfection reagent to transfect siRNA into renal cancer cells in proportion; S32: Knockdown efficiency verification: The knockdown efficiency of si-TMEM91 was detected by Western blotting WB, and the experiment was repeated to ensure the reproducibility and reliability of the results; S4: Functional experimental analysis; S41: Cell proliferation experiment, using MCE CCK-8 kit to evaluate cell proliferation ability; at different time points: 0h, 12h, 24h, 48h, measure absorbance and calculate cell proliferation rate; S42: Cell migration experiment, using transwell chamber to perform cell migration experiment; after culturing under appropriate conditions, the migration ability was analyzed by crystal violet staining and counting the cells that migrated to the lower layer under a microscope; S43: Cell apoptosis detection, apoptosis rate was analyzed by flow cytometry; cells were double stained with Annexin V / PI, and the proportion of apoptotic cells was analyzed by flow cytometry; S5: protein expression level analysis; S51: immunoblot analysis; S52: Result analysis.

2. The method for studying the effect of TMEM91 gene on renal clear cell carcinoma according to claim 1, characterized in that: In the above S1, the cell line is cultured in a cell incubator at 37° C. and containing 5% CO 2 , and the cell morphology and growth state are regularly observed to keep the cells in a good growth environment.

3. The method for studying the effect mechanism of TMEM91 gene on renal clear cell carcinoma according to claim 1, characterized in that: In the S21, the Flag-TMEM91 plasmid was constructed, including: TMEM91 sequence information was downloaded from NCBI, and CDS and restriction sites were analyzed using SnapGene software; Design specific primers and add homology arms, confirm the specificity through blast comparison, and then have BGI synthesize the primers; Using HEK 293T cell cDNA as a template, TMEM91 CDS was amplified by PCR, connected to the CMV-Flag vector, and purified plasmid was obtained by plasmid extraction.

4. The method for studying the effect of TMEM91 gene on renal clear cell carcinoma according to claim 1, characterized in that: In the S22, the cell transfection experiment includes: Flag-TMEM91 plasmid was transiently transfected into the target cell line using Lipofectamine2000; A control group and an experimental group were set up to conduct preliminary tests on transfection efficiency and gene expression levels, respectively.

5. The method for studying the effect mechanism of TMEM91 gene on renal clear cell carcinoma according to claim 1, characterized in that: The cell proliferation experiment is as follows: S411: Cell seeding: cells in good condition were digested with trypsin and counted, and seeded in a 96-well plate at a density of 1000 cells per well; at least 3 replicates were set for each experimental condition to ensure the accuracy of the data; S412: Cell attachment: Allow cells to grow in 96-well plates for 24 hours to ensure they are fully attached and proliferating before experimental treatment; S413: Experimental treatment, adding different concentrations of drugs, gene silencing or overexpression treatment; S414: Proliferation detection, using the MCE CCK-8 kit, CCK-8 solution was added to each well at a ratio of 1:100, and then incubated at 37°C for 1 hour; the absorbance value at 450nm was measured using an ELISA reader; measurements were performed at different time points after the start of the experiment: 0h, 12h, 24h, and 48h to evaluate cell proliferation.

6. The method for studying the effect of TMEM91 gene on renal clear cell carcinoma according to claim 5, characterized in that: The cell migration experiment is as follows: S421: Preparation of transwell chamber: Take a transwell chamber, the upper and lower chambers are separated by a polycarbonate membrane with a pore size of 8.0 μm; ensure that the chamber is sterile and clean; S422: Cell preparation and planting: resuspend the corresponding treated cells in serum-free medium and adjust the cell density to the appropriate concentration; add the cell suspension to the upper chamber, and add the medium containing 10% FBS as a chemical attractant to the lower chamber; S423: Cell culture, incubated at 37°C with 5% CO2 for 48 hours to allow sufficient time for cells to migrate through the polycarbonate membrane; S424: Fixation and staining: remove the upper chamber, wash with PBS to remove the non-migrated cells, then fix the cells that migrated to the bottom of the upper chamber with 4% paraformaldehyde, and stain the cells with 1% crystal violet solution. The staining time is adjusted according to the cell type and the strength of the staining solution; S425: Observation and analysis of results: Use a cotton swab to gently wipe off the non-migrated cells on the inner side of the upper chamber, then randomly select fields of view under a microscope to take pictures and count the migrated cells, and statistically analyze the cell migration ability.

7. The method for studying the effect of TMEM91 gene on renal clear cell carcinoma according to claim 6, characterized in that: The cell apoptosis detection is specifically as follows: S431: Cell collection: collect the cell culture medium from the culture plate and digest the cells with EDTA-free trypsin; mix the digested cells with the previously collected culture medium and collect the cells by gentle centrifugation; S432: Cell washing: wash the cells twice with cold PBS, discard the supernatant after centrifugation, and ensure that the cell pellet is intact; S433: Staining and analysis: resuspend the cells with staining buffer, add Annexin V and PI dyes according to the kit instructions, and incubate at room temperature in the dark for 15 minutes; analyze using flow cytometry to record the proportion of early apoptotic and late apoptotic cells.

8. The method for studying the effect mechanism of TMEM91 gene on renal clear cell carcinoma according to claim 1, characterized in that: The immunoblotting analysis comprises: After extracting total cell protein, the protein concentration was determined using a BCA kit; separation gel and concentration gel of corresponding concentrations were prepared according to the size of the target protein; After electrophoresis and membrane transfer, incubation was performed with specific antibodies, color was developed with developer, and the results were recorded using a gel imager.

9. The method for studying the effect mechanism of TMEM91 gene on renal clear cell carcinoma according to claim 1, characterized in that: The result analysis includes: The expression level changes of various proteins including TMEM91 under gene overexpression or silencing conditions were analyzed to analyze their biological functions in ccRCC.

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