Application of TMEM91 gene in diagnosis and treatment of cervical cancer

Abstract

The invention discloses application of a TMEM91 gene in diagnosis and treatment of cervical cancer, and relates to the technical field of biological medicines. The invention relates to shRNA (short hairpin Ribonucleic Acid) capable of specifically inhibiting the expression of a TMEM91 gene. The shRNA comprises a sequence 5 '-GCTGCAGTCTTCCAAGGATTC-3' or 5 '-GATGTTGAGGCATATGTCC-3', a sequence 5 '-GCTGCAGTAGGATTACC-3', a sequence 5 '- The shRNA is prepared by an annealing method and is used to reduce TMEM91 mRNA and protein levels. According to the invention, TMEM91 is determined as a cervical cancer treatment target; bioinformatics analysis and experimental verification show that TMEM91 is highly expressed in cervical cancer and is related to prognosis, the research of the TMEM family in cervical cancer is less, and particularly, the function of TMEM91 is not systematically reported; the invention fills the technical blank in the field, and provides a brand new target spot for cervical cancer treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of the TMEM91 gene in the diagnosis and treatment of cervical cancer. Background Technology

[0002] Cervical cancer is one of the most serious malignant tumors threatening women's health. As a global public health issue, cervical cancer poses a particularly heavy disease burden in low- and middle-income countries (LMICs).

[0004] Currently, in addition to traditional cytology examination (TCT), HPV testing has become an important screening method. Nevertheless, existing screening methods still have certain limitations. Therefore, exploring tumor molecular markers has significant potential for achieving early diagnosis and will also provide new ideas for cervical cancer screening and subsequent treatment. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing the application of the TMEM91 gene in the diagnosis and treatment of cervical cancer.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A shRNA that specifically inhibits TMEM91 gene expression, comprising the sequence 5'-GCTGCAGTTCTCCAAGGATTC-3' or 5'-GATGTTGAGGACATGTCATCC-3'; the shRNA is prepared by an annealing method and is used to reduce TMEM91 mRNA and protein levels.

[0007] A recombinant lentiviral vector containing the above-mentioned shRNA sequence is used to stably knock down TMEM91 expression.

[0008] A TMEM91 inhibitor, comprising a nucleic acid formulation containing the aforementioned shRNA.

[0009] A pharmaceutical composition comprising the above-described TMEM91 inhibitor and a pharmaceutically acceptable carrier; further comprising an autophagy regulator, said autophagy regulator being chloroquine or rapamycin.

[0010] A kit for detecting the interaction between TMEM91 and p53 proteins, comprising a TMEM91-specific antibody, a p53-specific antibody, and Protein A / G magnetic beads.

[0011] A method for preparing a TMEM91 knockdown cervical cancer cell model, characterized by comprising the following steps: The shTMEM91 sequence was designed and synthesized; a recombinant lentiviral vector was constructed; the virus was transfected into 293T cells; the virus was then used to infect cervical cancer cell lines; and stable strains were screened using puromycin.

[0012] A method for screening TMEM91 inhibitors, using the cell model described above; cell proliferation was detected by CCK-8 assay; and p53 protein levels were detected by Western blotting.

[0013] The use of shRNA as described above in the preparation of drugs for the treatment of cervical cancer.

[0014] The use of a TMEM91 inhibitor as described above in the preparation of drugs that regulate p53 protein levels via the autophagy pathway.

[0015] The beneficial effects of this invention are as follows: 1. This invention identifies TMEM91 as a therapeutic target for cervical cancer. Through bioinformatics analysis and experimental verification, it was found that TMEM91 is highly expressed in cervical cancer and is associated with prognosis. Previous studies on the TMEM family in cervical cancer were limited, especially the function of TMEM91, which has not been systematically reported. This invention fills a technological gap in this field and provides a novel target for the treatment of cervical cancer.

[0016] 2. This invention elucidates the TMEM91-p53 autophagy degradation pathway at multiple levels; a series of experiments have confirmed that TMEM91 and p53 have a direct interaction and regulate p53 degradation through autophagy; the mechanism study is in-depth, with solid data supporting both molecular interactions and pathway regulation. Attached Figure Description

[0017] Figure 1 This is a schematic diagram illustrating how overexpression or knockdown of TMEM91 affects the proliferation, migration, and apoptosis of cervical cancer cells, as presented in this invention. Figure 2 This is a schematic diagram illustrating the role of TMEM91 in regulating autophagy in this invention; Figure 3 This is a schematic diagram illustrating the interaction between TMEM91 and p53 in this invention, and how it affects the protein expression of p53. Figure 4 This is a schematic diagram of the degradation of p53 by TMEM91 via autophagy in this invention. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0019] Example 1: Application of TMEM91 gene in the diagnosis and treatment of cervical cancer, the specific scheme is as follows: 1. Materials and Methods 1.1 Material Preparation 1.1.1 Strains, cell lines, plasmids, siRNA, mice Cell lines: human embryonic kidney epithelial cell line HEK293T, human cervical cancer cell line Caski, and human cervical cancer cell line Hela.

[0020] Strains: competent cells DH5α; Plasmids FC-TMEM-91, GFP-TMEM91, and Sh-TMEM91 were all constructed by ourselves.

[0021] 1.1.2 Preparation of Main Reagents and Consumables 1.1.3 Preparation of Instruments and Equipment 1.1.4 Preparation of main reagent formulations, as detailed below: SDS-PAGE separation solution: 4% SDS-PAGE separation solution: 1.2 Methods 1.2.1 Plasmid Construction (1) Primer design 1) Access the NCBI Gene database and search for the human gene TMEM91 (NM_001098821). Locate and select the longest protein-coding transcript of this gene, obtain its CDS (Coding Sequence) sequence, and save it as a .fasta file. Open the CDS sequence using SnapGene. Based on the multiple cloning site of the selected cloning vector, add specific restriction endonuclease recognition sequences and necessary protective bases to the 5' end of the primers. Use the NCBIBLAST tool to align the designed primer sequences to ensure high specificity in the human genome and no non-specific amplification. Finally, add homologous arms. Send the finalized primer sequences to BGI Genomics for synthesis. The specific sequences are as follows: TMEM91 Forward: 5'-ATGGACAGCCCTAGTCTTCGT-3' TMEM91 Reverse: 5'-GCCTCCCGAGACCCGCCCTAG-3' 2) shRNA targeting TMEM91 was designed using the website (https: / / www.sigmaaldrich.cn / CN / zh), and primers were synthesized by BGI Genomics. The sequence is as follows: shTMEM91 ①:5'-GCTGCAGTTCTCCAAGGATTC-3' shTMEM91 @: 5'-GATGTTGAGGACATGTCATCC-3'shRNA oligo primer annealing: Prepare the following annealing system in a sterile PCR tube or centrifuge tube: oligo primer annealing system: Place the mixture in a PCR instrument and run the following program: 95℃, 5 min, then slowly cool to room temperature.

[0022] (2) Extraction of total RNA from cells 1) Discard the culture medium from the 6-well plate and gently wash the cells three times with pre-cooled PBS. Add 175 μL of lysis buffer (containing β-mercaptoethanol) to each well and gently shake horizontally to cover the entire well with the lysis buffer. Scrape off the cells with a cell scraper and transfer all the viscous cell lysis buffer to an RNase-free 1.5 mL centrifuge tube.

[0023] 2) Add 350 μL LRD Buffer to the lysis buffer and immediately mix vigorously by inverting. Incubate the mixture in a 70°C metal bath for 5 min, then briefly cool on ice. Centrifuge at 4°C, 13,000 rpm for 10 min. Carefully aspirate the supernatant (avoiding precipitate) and transfer it to a new RNase-free tube.

[0024] 3) Add 200 μL of anhydrous ethanol to the supernatant and gently invert to mix. Transfer the entire mixture to an RNA purification column, centrifuge at 13,000 rpm for 2 min at 4 °C, and discard the filtrate. Prepare the DNase I working solution (40 μL DDNase Buffer + 9 μL 25 mMnCl2 + 1 μL DDNase I), mix well, and add directly to the center of the purification column matrix. Incubate at room temperature for 15 min. Add 200 μL DNaseStop Buffer, centrifuge at 12,000 rpm for 30 seconds at 4 °C, and discard the filtrate.

[0025] 4) Add 600 μL Wash Buffer, centrifuge at 12,000 rpm for 30 seconds at 4°C, and discard the filtrate. Repeat this washing step once. Centrifuge the empty column at 12,000 rpm for 2 minutes at 4°C to completely remove residual ethanol. Transfer the purified column to a new RNase-free collection tube, add 30 μL of Efficacy Buffer to the center of the column matrix, and incubate at room temperature for 5 minutes. Collect RNA by centrifuging at 12,000 rpm for 1 minute at 4°C.

[0026] 5) Use NanoDrop2000 to detect RNA quality, record the absorbance values ​​(OD values) at 260nm and 280nm, as well as the RNA concentration, and assess purity (A260 / A280 ratio should be between 1.8 and 2.1). After passing the test, the RNA solution should be used immediately for reverse transcription, or aliquoted and stored at -80℃, avoiding repeated freeze-thaw cycles.

[0027] (3) Reverse transcription 1) Calculate the required volume of RNA solution based on the concentration measured by NanoDrop2000. Prepare the following mixture in a PCR tube: Reverse transcription system I: 2) Gently pipette to mix, then briefly centrifuge. Place the mixture in a PCR instrument and incubate at 65°C for 5 minutes. Immediately after incubation, transfer the PCR tube to an ice pack and allow it to cool for 5 minutes before adding the reagents for the second step: Reverse transcription system II: 3) After mixing thoroughly by pipetting with the tip of a pipette and briefly in vitro, place the PCR tube back into the PCR instrument to complete the second step of the reverse transcription reaction procedure: Reverse transcription procedure: 4) After the reaction is complete, store the cDNA product at -80°C.

[0028] (4) PCR amplification to obtain the target fragment 1) Using a high-fidelity DNA polymerase (such as KOD-plus-neo), cDNA is used as a template and PCR amplification is performed through specific primers to obtain a high-fidelity target gene fragment for subsequent vector construction.

[0029] PCR reaction system: 2) Gently pipette the reaction mixture to mix thoroughly. After a brief separation, place the PCR tube in the PCR instrument and start the following PCR program: PCR procedure: 3) Weigh an appropriate amount of agarose powder according to the size of the electrophoresis tank, and add an appropriate amount of 1×TAE buffer. Heat intermittently in a microwave oven until the agarose is completely dissolved and the solution is clear. Cool the gel solution until it feels warm to the touch but not hot, add the nucleic acid dye according to the recommended ratio in the instructions, and mix gently. Pour the solution into the gel casting tank with the comb inserted, and let it stand at room temperature for 15-20 minutes until it is completely solidified.

[0030] 4) After the PCR program is complete, take 5 μL of PCR product and mix it thoroughly with 1 μL of 6× DNA Loading Buffer to achieve a final loading buffer concentration of 1× working concentration. Place the solidified gel into the electrophoresis tank and add sufficient fresh 1× TAE buffer, ensuring the liquid level completely covers the gel surface by approximately 1-2 mm. Carefully remove the comb vertically upwards. Add the following to the gel wells in sequence: DNA Marker (e.g., 5 μL) and the prepared PCR product sample. Cover the electrophoresis tank, connect the power supply, set it to a constant voltage of 120V, and electrophoresis for approximately 25 minutes.

[0031] 5) After electrophoresis, turn off the power and remove the gel. Place the gel in a gel imaging system and expose it to UV light for imaging. By comparing the positions of the DNA marker bands, confirm the expected molecular weight and specificity of the target band (whether it is a single bright band). Using a clean blade or gel cutter, quickly and accurately cut off the gel block containing the target band under UV light. Transfer the gel block to a pre-weighed 1.5mL centrifuge tube, weigh it again, and record the net weight of the gel block for subsequent use in the DNA gel recovery and purification kit.

[0032] (5) Glue recycling: 1) Based on the weight of the gel block, add the corresponding volume of sol solution at a ratio of m / v=1:1 (i.e., 1 mg of gel to 1 μL of sol solution).

[0033] 2) Place the centrifuge tube in a 65°C metal bath and invert it intermittently to mix until the gel block is completely dissolved.

[0034] 3) Transfer all the solution to the adsorption column provided in the kit and let it stand at room temperature for 5 minutes. Place the adsorption column in a centrifuge tube, centrifuge at 12,000 rpm for 60 seconds, and discard the filtrate in the collection tube.

[0035] 4) Add an appropriate amount of washing solution to the adsorption column, let it stand for 5 minutes, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate.

[0036] 5) Repeat step 4) once for the second wash to thoroughly remove impurities.

[0037] 6) Return the empty adsorption column to the collection tube and centrifuge at 12,000 rpm for 2 min to completely remove residual ethanol.

[0038] 7) Place the adsorption column in a new sterile centrifuge tube and add 30 μL of preheated (65°C) elution buffer or sterile ddH2O to the center of the adsorption membrane.

[0039] 8) Let stand at room temperature for 2 minutes to allow the eluent to fully wet the adsorption membrane.

[0040] 9) Centrifuge at 12,000 rpm for 2 minutes. The liquid in the centrifuge tube is the recovered DNA product. To increase the yield, the effluent can be added back to the adsorption membrane and centrifuged again.

[0041] 10) The concentration and purity were determined using a Nanodrop 2000 (A260 / A280≈1.8-2.0). The product was stored at -20℃ for later use.

[0042] (6) Enzyme digestion of vector 1) Prepare the following reaction system in a sterile PCR tube on ice: Double enzyme digestion system (taking a 20μL total volume as an example): 2) Place the reaction tube in a 37°C water bath and incubate for 4 hours. For complex vectors or large amounts of DNA, overnight incubation (12-16 hours) is possible, in which case the amount of enzyme should be reduced accordingly.

[0043] 3) After enzyme digestion, heat the reaction tube at 65℃ for 10 minutes to inactivate the enzyme. Agarose gel electrophoresis is then performed to recover the enzyme, simultaneously verifying complete digestion and purifying the target linearized vector. A small amount of the product before and after purification is taken for agarose gel electrophoresis to confirm complete digestion (no circular vector residue).

[0044] 4) The concentration and purity of the purified linear vector were determined using a Nanodrop 2000 (A260 / A280≈1.8-2.0). The purified linearized vector was aliquoted and stored at -20℃ to avoid repeated freeze-thaw cycles.

[0045] (7) LIC connection 1) Use exonuclease III (ExoIII) to ligate the target fragment and the linearized vector, as follows: LIC connection system: 2) Gently pipette the reaction mixture several times to ensure thorough mixing. Place the reaction tube on ice for 5 minutes. Add 1 μL of LExoIII (20 U / μL) to the reaction mixture. After briefly centrifuging to collect the liquid at the bottom of the tube, continue to place the reaction tube on ice for 1 hour. After the reaction is complete, add 1 μL of 0.5 MEDTA (pH 8.0) to terminate the enzymatic digestion. Then transfer the entire reaction mixture to a 65°C metal bath and incubate for 5 minutes to ensure complete heat inactivation of the enzyme.

[0046] (8) T4 connection 1) The following is a 10 μL ligation reaction system, prepared entirely on ice: T4 connection system: 2) Place the reaction tube in a PCR instrument at 16℃ and incubate for 4-16 hours (overnight is best). After ligation, the reaction tube can be heated at 65℃ for 10 minutes to heat-inactivate the ligase. The product can be used immediately for transformation.

[0047] (9) Transformation 1) Remove competent cells (DH5α) from the -80℃ freezer beforehand and immediately place them on ice to thaw. Once the cells are completely thawed into an ice-water mixture, gently tap the tube wall to mix. Transfer 50 μL of competent cells to a sterile centrifuge tube. Add 10 μL of the ligation product to be transformed and gently stir with a pipette tip to mix; do not vigorously pipette. Let the mixture stand on ice for 30 minutes.

[0048] 2) Quickly transfer the centrifuge tubes to a 42°C constant temperature water bath, heat shock for 45 seconds, and then immediately place the centrifuge tubes back on ice and let them stand for 2-5 minutes.

[0049] 3) Add 600 μL of pre-warmed LB medium (antibiotic-free) to the tube. Place the centrifuge tube in a 37°C shaker and incubate at 200 rpm for 60 min to allow the cells to recover and express the antibiotic resistance gene.

[0050] 4) Briefly centrifuge the revived bacterial culture (6000 rpm, 2 min), discard some of the supernatant, and retain approximately 100 μL to resuspend the bacteria. Add all the bacterial culture to an LB agar plate containing the appropriate antibiotic and spread it evenly with a sterile spreader. After the liquid on the surface of the plate has been absorbed, invert the plate and incubate at 37°C for 12-16 hours (overnight).

[0051] (10) Colony PCR 1) Preparation of bacterial suspension (template): Near an alcohol lamp, use a sterile pipette tip to pick up a single colony with regular shape and good dispersion from the transformation plate. Suspend the bacterial cell in 10 μL of sterile ddH2O and gently pipette to mix. This is the colony PCR template.

[0052] 2) Prepare the reaction system (total volume 10 μL) on ice according to the following proportions: Colony PCR system: 3) Primer design: The upstream primer is selected from the universal sequence on the vector backbone, and the downstream primer is selected from the specific sequence of the inserted target gene to ensure that the amplification product can clearly distinguish between the empty vector and the recombinant vector.

[0053] 4) Gently tap the tube wall to mix the system, briefly separate it, and then place it in the PCR instrument to run the standard procedure: Colony PCR procedure: (11) Plasmid extraction: 1) Pour the overnight culture into a centrifuge tube and centrifuge at 4000 rpm for 10 minutes at 4°C. Discard the supernatant completely and retain the bacterial pellet.

[0054] 2) Add 500 μL of Buffer P1 to the precipitate, and resuspend thoroughly by pipetting to ensure no clots form. Let stand at room temperature for 5 min. 3) Add 500 μL of Buffer P2, and immediately and gently invert the centrifuge tube 6-8 times until the solution becomes viscous and clear. Let stand at room temperature for 5 minutes.

[0055] 4) Add 500 μL Buffer P3, and immediately and gently invert the centrifuge tube 6-8 times. At this point, a large amount of white flocculent precipitate will appear. Let stand at room temperature for 5 minutes, then centrifuge at 13,000 rpm for 15 minutes.

[0056] 5) Assemble the endotoxin-free filter column. After centrifugation, carefully aspirate the supernatant and transfer it to the filter column in portions (≤750 μL each time). Centrifuge at 13000 rpm for 1 min, and collect the filtrate in a new 2 mL EP tube.

[0057] 6) Add 450 μL of isopropanol to the filtrate, mix by turning the container upside down 6-8 times, and let it stand at room temperature for 2 minutes.

[0058] 7) Add 200 μL of Buffer PS to the DNA adsorption column, centrifuge at 13000 rpm for 2 min, and discard the waste liquid.

[0059] 8) Add the mixture from step 6 in portions (≤750μL each time) to the activated adsorption column, centrifuge at 13000rpm for 1min, discard the waste liquid, and continue until all the solution passes through the column.

[0060] 9) Add 750 μL of Buffer PW to the adsorption column, let stand at room temperature for 5 min, centrifuge at 13000 rpm for 1 min, and discard the waste liquid. Add another 750 μL of Buffer PW, no need to let stand, centrifuge at 13000 rpm for 1 min, and discard the waste liquid.

[0061] 10) Replace the adsorption column with an empty collection tube and centrifuge at 13,000 rpm for 2 minutes to completely remove residual ethanol.

[0062] 11) Elution: Transfer the adsorption column to a new sterile, enzyme-free 1.5 mL EP tube. Open the cap of the adsorption column and allow it to air dry in a laminar flow hood for about 5 minutes to allow residual ethanol to evaporate completely.

[0063] 12) Add 100-200 μL of preheated BufferEB (preheating at 65℃ can improve efficiency) to the center of the adsorption membrane and let it stand at room temperature for 2 min. Centrifuge at 13000 rpm for 2 min and collect the liquid at the bottom of the tube, which is the high-purity plasmid DNA.

[0064] 1.2.2 Cell Culture (1) Cell resuscitation 1) Quickly remove the cryovials from the liquid nitrogen tank or -80°C freezer, immediately place them in plastic film gloves, put them in a 37°C constant temperature water bath, gently shake to thaw completely within 2 minutes, then centrifuge at 1000 rpm. 3 min, prepare a 15 ml centrifuge tube in advance and add 4 ml of complete culture medium.

[0065] 2) After centrifugation, discard the cryopreservation solution. Mix the cells with 1 ml of complete culture medium by pipetting, then transfer the cell suspension to a pre-prepared sterile 15 ml centrifuge tube, centrifuge at 1000 rpm for 3 min, and discard the supernatant.

[0066] 3) Resuspend the cell pellet in 1 mL of complete culture medium. Transfer to a 6 cm culture dish and add 3 mL of complete culture medium. Mix by pipetting and then by cross-mixing to ensure the cells are evenly distributed in the culture dish. After culturing for 24 hours, discard the old culture medium (to remove floating dead cells) and add fresh complete culture medium.

[0067] (2) Cell passage 1) Passage is performed when the cell confluence reaches 80-90%.

[0068] 2) Discard the old culture medium. Add 1-2 mL of sterile 1×PBS solution, gently agitate to wash the cell surface, and discard the PBS. Add an appropriate amount of trypsin digestion solution, gently agitate to evenly cover the cells, and place in an incubator for digestion. Observe under a microscope; when the intercellular spaces increase, the cells become rounded, and some cells detach, immediately add an equal volume of complete culture medium to stop the digestion.

[0069] 3) Gently pipette the bottom of the dish to completely detach the cells, forming a single-cell suspension. Transfer the suspension to a centrifuge tube, centrifuge at 1000 rpm for 3 minutes, and discard the supernatant. Resuspend the cells in an appropriate amount of complete culture medium. Inoculate the cells into new culture dishes at the required passage ratio (e.g., 1:3 or 1:4) and replenish with sufficient culture medium. Clearly label the cell line name, passage date, and passage number.

[0070] 4) Experiments can only be conducted after the cells have grown stably for 2-3 generations and recovered to a good condition.

[0071] (3) Cell cryopreservation.

[0072] 1.2.3 Lentiviral-mediated construction of stable cell lines (1) First, select the vector. Overexpression: Select a lentiviral overexpression plasmid containing a strong promoter (CMV). Gene silencing: Select an shRNA expression plasmid containing a U6 promoter.

[0073] (2) Constructing recombinant plasmids: clone the target gene fragment (cDNA, shRNA sequence, etc.) into the selected lentiviral vector and perform sequencing verification.

[0074] (3) Plate healthy 293T cells to a suitable density (approximately 70%-80% confluence). Use a transfection reagent (such as PEI) to co-transfect the three plasmids (psPAX2, pMD2.G, and target plasmid) into the 293T cells at an optimized ratio (usually target plasmid:psPAX2:pMD2.G=4:3:1).

[0075] (4) Replace the medium with 20% FBS 24 hours after plasmid transfection. Collect the filtered viral supernatant 48 hours after transfection through a 0.45 μm filter membrane to remove cell debris.

[0076] (5) Plate the target cells to a suitable density (usually 30%-50%). Infect the cells with the above-mentioned virus solution and complete culture medium containing Polybrene (usually a final concentration of 6-8 μg / mL). Replace with fresh complete culture medium 8-12 hours after infection.

[0077] (6) 48 hours after infection, the culture medium was replaced with one containing the optimal selection concentration of antibiotic (purinomycin). Screening continued for 7-14 days until all uninfected control group cells died and the experimental group formed obvious resistant clones.

[0078] (7) Microscopic observation: If the vector carries a fluorescent reporter gene (such as GFP, RFP), the fluorescence expression can be observed directly. Alternatively, qPCR / Western Blot can be used to detect the expression of the target gene at the mRNA and protein levels.

[0079] 1.2.4 Transient transfection of cells (1) Plasmid transfection 1) About 24 hours before transfection, digest and count the cells in the logarithmic growth phase, and spread them evenly in the culture plate / dish at an appropriate density.

[0080] 2) The cell confluence during transfection should be 70%-90% (most commonly 70%-80%), and the specific optimal density varies depending on the cell type.

[0081] 3) Transfection complex preparation (using a 6-well plate as an example, scale up or down proportionally): Transfect 3 µg of plasmid into each well. Take a sterile centrifuge tube (e.g., a 1.5 mL EP tube) and add 100 µL of serum-free, antibiotic-free basal medium (e.g., Opti-MEM, or serum-free medium specifically for this cell type). Add the calculated weight of plasmid DNA to this medium and mix gently.

[0082] 4) Take another sterile centrifuge tube and add an equal volume of 100µL of the same serum-free basal medium. According to the starting ratio recommended in the reagent instructions (e.g., for Lipofectamine 2000, DNA (µg): reagent (µL) = 1:3), add the corresponding volume of transfection reagent to the medium, gently pipette or vortex to mix, and let stand at room temperature for 5 minutes.

[0083] 5) Add the diluted transfection reagent solution to the DNA-containing solution. Immediately mix gently by pipetting or vortexing. Let stand at room temperature for 10 minutes to allow the DNA to fully bind with the transfection reagent and form a stable, positively charged nanoscale complex.

[0084] 6) Add 200µL of the prepared transfection complex dropwise and evenly to each well, gently shaking the culture plate while adding to ensure even distribution. Return the cells to a 37°C, 5% CO2 incubator for further culture.

[0085] 7) 4-6 hours after transfection (or 6-24 hours as per instructions), the culture medium containing the complex must be aspirated and replaced with fresh, complete culture medium containing serum and antibiotics.

[0086] 8) Exogenous protein expression usually reaches its peak at 48-72 hours, which is the optimal time to collect samples for Western Blot, qPCR, immunofluorescence, or luciferase reporter gene detection.

[0087] 1.2.5 Quantitative Real-Time PCR 1) The quantitative PCR primer sequences used in this experiment were obtained from the Primer Bank database or the Origine online design tool. All primer sequences were specifically validated using the NCBI Primer-BLAST tool.

[0088] The specific primer sequences are as follows: TMEM91 qPCR Forward:5'-GTGACAGTGACTCGGACTGG-3' TMEM91 qPCR Reverse:5'-CCAAGCCTTGTTGGTCTCTG-3' p53 qPCR Forward:5'-AAGTCTAGAGCCACCGTCCA-3' p53qPCR Reverse:5'-GACACTCCTCAGCCCCAATC-3' GAPDH qPCR Forward:5'-TCGGAGTCAACGGATTTGGT-3' GAPDH qPCR Reverse:5'-TTCCCGTTCTCAGCCTTGAC-3' 2) Dilute the cDNA template synthesized by reverse transcription 5-fold with sterile nuclease-free water, mix well, briefly centrifuge, and then place on ice for later use. Perform real-time quantitative PCR using the SYBR Green dye method. Three technical replicates were set up for each sample. The entire experiment was conducted in a nuclease-free environment, and pipette tips with filters were used to prevent contamination.

[0089] RT-qPCR reaction components and dosages: RT-qPCR reaction execution procedure: 3) Using the GAPDH gene as an internal control, the relative expression levels of the target genes (TMEM91, p53, VAMP8, SNAP29, STX17) were calculated using the 2ΔΔCt method. First, the difference in Ct values ​​(ΔCt) between the target gene and the internal control gene for each sample was calculated. Then, using the mean ΔCt of the control group samples as the calibration benchmark, the ΔΔCt values ​​of the experimental groups were calculated. The final relative expression level was expressed as 2ΔΔCt.

[0090] 1.2.6 CCK8 cell proliferation experiment (1) Cell plating: Take cells in the logarithmic growth phase, digest and resuspend them, and adjust the cell density with complete culture medium.

[0091] (2) Seed 100 μL of cell suspension into each well of a 96-well plate. The number of cells to be seeded needs to be determined through preliminary experiments. Generally, it is advisable to keep the absorbance of the control group between 0.8 and 1.2 at the end of the experiment.

[0092] Experimental well setup: A. Blank control: Culture medium and cell-free CCK-8 reagent (for instrument zero-point calibration). B. Control group: Culture medium and CCK-8 reagent for stable overexpression cells (GFF-CTL) and stable knockdown cells (sh-CTL). C. Experimental group: Culture medium and CCK-8 reagent for stable overexpression cells (GFP-TMEM91) and stable knockdown cells (sh-TMEM91).

[0093] (3) Place the culture plate in a 37℃, 5% CO2 incubator for a period of time until the cells are completely attached to the plate.

[0094] (4) After reaching the predetermined time point, discard the old culture medium. Add a mixture of 10 μL CCK-8 reagent and 100 μL blank culture medium to each well. Avoid generating air bubbles during sample addition to prevent affecting the absorbance reading. Place the culture plate back in the incubator and incubate in the dark for 1.5 hours. (The optimal incubation time was determined through preliminary experiments; an OD450 value between 0.8 and 1.2 indicates the optimal incubation time.) (5) After incubation, remove the 96-well plate from the incubator. Use a microplate reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0095] (6) Calculate the average OD values ​​of the blank control group, the control group, and each experimental group. Calculate cell viability using the OD values.

[0096] 1.2.7 Cell Scratch Assay (1) Digest and resuspend the cells, count them, and then seed them into 6-well plates.

[0097] (2) Once the cells have fully merged, use a 200 μL sterile pipette tip (or a dedicated scratcher) perpendicular to the bottom of the well plate and steadily draw a straight line along a ruler or marker line. Discard the old culture medium and gently rinse the well plate three times with PBS buffer to remove the scratched cell debris.

[0098] (3) Immediately after scratching (0 hours), use an inverted microscope under a 4x objective lens to take pictures of 3-5 fixed sites in the scratched area. Then, take pictures of the same sites every 24 hours until the scratches in the control group are basically closed.

[0099] (4) Data analysis: Image analysis software such as ImageJ is used to measure the width or area of ​​the scratch at each time point.

[0100] 1.2.8 Apoptosis (1) Take the cell suspension and seed it in a 6-well plate. After the cell density reaches 80%-90%, proceed with the subsequent experiments.

[0101] (2) Use trypsin without EDTA for digestion, and collect the cell supernatant (which may contain detached apoptotic cells) into a centrifuge tube. Centrifuge at 1000 rpm for 5 min at room temperature, and discard the supernatant.

[0102] (3) Gently resuspend the cell pellet in pre-chilled PBS and centrifuge again. Repeat this step 1-2 times to thoroughly remove proteins from the culture medium. Discard the supernatant, add an appropriate amount of pre-chilled PBS, resuspend and count the cells, adjusting the cell density to 1×10⁻⁶. 6 ~5×10 6 Cells / mL. Cell apoptosis was detected at the flow cytometry platform of the School of Life Sciences, Chongqing Medical University.

[0103] 1.2.9 Immunoblotting (1) Protein extraction: Discard the culture medium and wash the cells with pre-cooled PBS. Add an appropriate amount of 1% SDS and scrape off the cells with a cell scraper, collecting the lysis buffer into a centrifuge tube. Boil in a 95°C metal bath for 10 min, then centrifuge at 1,3000 rpm for 10 min. Transfer the supernatant to a new tube (do not aspirate the cell pellet), and determine the protein concentration using the BCA method.

[0104] (2) Protein concentration determination by BCA method: Take 2 mg / mL of BSA standard stock solution. Use the same solvent as the sample to be tested to serially dilute it to 8 concentrations: 0, 0.25, 0.5, 0.75, 1.0, 1.5, and 2.0 mg / mL. After aliquoting, store at -20℃ for a short period to avoid repeated freeze-thaw cycles. Calculate the required total amount of BCA working solution based on the quantity of the sample to be tested (including standards) and the volume ratio of reagent A to reagent B = 50:1. Prepare sterile 1.5 mL centrifuge tubes and add samples according to the following system: Standard wells: 95 μL BCA working solution + 5 μL of standards at each concentration. Sample wells: 98 μL BCA working solution + 2 μL of the protein sample to be tested. Vortex or gently pipette to mix the contents of each tube. Cap the tubes and incubate in a 37℃ water bath for 30 min in the dark.

[0105] (3) Preparation of SDS-PAGE gel: Assemble the plate: Align the clean, dry glass plate and gasket, and correctly place them in the gel casting holder to ensure a leak-free bottom seal. Select and calculate the optimal concentration of the separating gel according to the molecular weight of the target protein. Prepare the separating gel: Mix the gel casting reagent according to the SDS-PAGE separating solution formula. Quickly pour the mixed separating gel solution into the mold along one side of the glass plate, filling it to about 2-2.5 cm from the upper edge of the short glass plate. Then, gently cover the gel solution with a layer of isopropanol using a pipette to seal it, removing air bubbles and isolating it from air, making the gel surface smooth. Let it stand vertically at room temperature for 20-30 minutes. After the gel has completely polymerized (a clear dividing line is visible), pour off the upper sealing solution, gently rinse the gel surface along the glass plate 2-3 times with ultrapure water, and carefully absorb any remaining water stains with filter paper. Prepare the stacking gel: Mix the gel casting reagent according to the 4% SDS-PAGE separating solution formula. Pour the concentrated gel solution directly onto the solidified separating gel until the liquid overflows from the top of the short glass plate. Then, tilt the 1.5mm comb at a certain angle and slowly insert it into the liquid from one side, carefully adjusting it to a horizontal position to ensure that the bottom of the comb is completely immersed in the gel solution without any air bubbles remaining. Let it stand vertically at room temperature for 15-20 minutes to allow the concentrated gel to fully polymerize.

[0106] (4) Protein loading: Mix the protein sample with 1% SDS lysis buffer in the correct ratio, then add loading buffer to make up the difference. Boil in a 95°C metal bath for 10 min to fully denature the protein. The sample can then be loaded immediately or stored at -80°C. Place the gel plate into the electrophoresis tank and add 1× electrophoresis buffer. Gently remove the comb. Add protein marker and an equal amount of total protein sample to the wells.

[0107] (5) Electrophoresis: The initial voltage is set to 80V. After the sample enters the separating gel and the bromophenol blue forms a straight line, the voltage is switched to 120V and electrophoresis is continued until the bromophenol blue reaches the bottom of the gel.

[0108] (6) Transfer: Prepare transfer buffer in advance and place on ice. Cut the PVDF membrane to the size of the gel and activate it by soaking in methanol for 15 seconds. Then, soak the membrane, filter paper, and sponge in the transfer buffer. Make a "sandwich": place the membrane in the transfer clamp in the following order: cathode → sponge → filter paper → gel → PVDF membrane → filter paper → sponge → anode. Each step requires precise alignment and removal of all air bubbles. The operation must be performed in the transfer buffer. Place the transfer clamp in the transfer tank, add the pre-cooled transfer buffer, and place the entire apparatus in an ice-water mixture. Set the electrophoresis current to 300mA for constant current transfer.

[0109] (7) Blocking: After the transfer is completed, the PVDF membrane is taken out and placed in a blocking solution containing 5% skim milk TBST, and blocked on a shaker at 4°C for 4 hours.

[0110] (8) Incubation of primary antibody: Dilute the primary antibody with blocking agent according to the ratio recommended in the instructions. Incubate the membrane with the primary antibody solution on a shaker at 4°C overnight; recover the primary antibody and wash the PVDF membrane three times with TBST on a shaker at room temperature for 8 min each time.

[0111] (9) Incubation with secondary antibody: Dilute the HRP-labeled secondary antibody with blocking buffer according to the specified ratio. Incubate the membrane with the secondary antibody solution on a shaker at room temperature for 1-2 hours. Wash the PVDF membrane again with TBST on a shaker at room temperature 4 times, 5 minutes each time, to completely remove unbound secondary antibody.

[0112] (10) Development: According to the instructions of the chemiluminescence kit, mix equal volumes of substrate solutions A and B. Use filter paper to remove excess TBST from the membrane, and evenly drop the mixed luminescent substrate onto the membrane. React for 1-2 minutes. Use filter paper to remove excess substrate solution, and place the membrane in a chemiluminescence imager for image acquisition and data analysis.

[0113] 1.2.10 Immunoprecipitation (1) Seed healthy 293T cells into 6cm culture dishes. When the cell confluence reaches 60%-70%, use a suitable transfection reagent to transfect the target plasmid with the CMV-FC / GFP tag. Eight hours after transfection, replace with fresh complete culture medium and continue culturing for 48 hours to ensure full expression of the target protein.

[0114] (2) Forty-eight hours after transfection, gently wash the cells three times with pre-chilled PBS. Add 1 mL of pre-chilled RM lysis buffer to each dish, and collect the cell lysate into pre-chilled enzyme-free EP tubes using a cell scraper. Incubate on ice for 30 min, and sonicate the lysis buffer until the solution is no longer viscous and becomes clear. Centrifuge at 1,3000 rpm for 15 min at 4°C. Transfer the supernatant to a new pre-chilled EP tube and place on ice.

[0115] (3) Take a small amount of cell lysis supernatant and determine the protein concentration using the BCA method.

[0116] (4) Take an appropriate amount of Protein A / G (anti-GFP) magnetic bead suspension (20 μL / sample recommended) and transfer it to a pre-chilled 1.5 mL enzyme-free EP tube. Add 1 mL of pre-chilled 0.05% Tween-20 PBST buffer and gently resuspend the magnetic beads. Centrifuge at 2000 rpm for 3 min at 4 °C and carefully discard the supernatant. To completely remove residual liquid, you can briefly centrifuge at high speed and then aspirate it with a small-range pipette. Repeat this washing step for a total of 3 times. For the last time, resuspend the washed magnetic beads with 1 mL of RM lysis buffer and aliquot them into each EP tube according to the number of samples. Place them on ice for later use.

[0117] (5) Based on the concentration calculation, take the volume of lysis buffer equivalent to 1 mg of total protein and add it to the EP tube containing the pretreated magnetic beads. Make up the reaction system to 1 mL with RM lysis buffer to ensure that the lysis buffer and magnetic beads are in full contact. Place the EP tube on a 4°C rotating shaker and incubate gently overnight.

[0118] (6) The next day, centrifuge at 2000 rpm for 3 min at 4℃, carefully discard the supernatant, avoiding aspirating the magnetic beads. Add 1 mL of pre-chilled RM lysis buffer to the tube and resuspend the magnetic beads. Place the EP tube on a 4℃ rotary shaker and wash at 35 rpm for 5 min. Repeat this washing step for a total of 4 times to thoroughly remove non-specifically bound impurities. After the last wash, thoroughly aspirate the supernatant. Add 40 μL of 1×SDS-PAGE loading buffer to the precipitate and vortex to mix. Heat at 95℃ for 10 min to denature the protein. Centrifuge at 12000 rpm for 3 min at room temperature and collect the supernatant. This supernatant is the immunoprecipitate product, which can be used directly for Western blotting experiments or temporarily stored at -20℃.

[0119] 1.2.11 Statistical Analysis All experiments in this study were independently repeated at least three times. Data are expressed as mean ± standard deviation (Mean ± SD). Statistical tests were performed using the independent samples t-test and two-way ANOVA. If the ANOVA results showed significant differences, post-hoc tests were used for inter-group comparisons. The statistical significance level was set at α = 0.05, and the following notations were used to represent the significance level of differences: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0120] 2. Results 2.1 Overexpression or knockdown of TMEM91 affects the proliferation, migration, and apoptosis of cervical cancer cells. To further explore the biological function of TMEM91 in cervical cancer, we constructed cell models with stable overexpression or knockdown of TMEM91 and conducted a series of phenotypic experiments. First, we stably overexpressed GFP-labeled TMEM91 in HeLa cells. Cell scratch assays, CCK-8 assays, and colony formation assays showed that overexpression of TMEM91 significantly promoted the migration of HeLa cells. Figure 1 A, C) and proliferation capacity ( Figure 1 E, G, I). Furthermore, Annexin V-FITC / PI double staining flow cytometry analysis showed that overexpression of TMEM91 inhibited apoptosis (E, G, I). Figure 1 K, M).

[0121] To verify the generalizability of this function, we transformed the Caski cell line to construct a cell model with stable TMEM91 knockdown. Functional recovery experiments showed that TMEM91 knockdown effectively inhibited Caski cell migration. Figure 1 B, D) and proliferation ( Figure 1 F, H, J), and promote their apoptosis (F, H, J), Figure 1 L, N).

[0122] To further elucidate the molecular mechanism by which TMEM91 regulates apoptosis, we used Western blotting to detect changes in the expression of key apoptosis-related proteins. The results showed that overexpression of TMEM91 in HeLa cells significantly downregulated the expression of the pro-apoptotic protein Bax, while upregulating the expression of the anti-apoptotic protein Bcl-2. Conversely, knockdown of TMEM91 in Caski cells led to increased Bax expression and decreased Bcl-2 expression. Figure 1 O).

[0123] In summary, these experimental results indicate that TMEM91 can promote the proliferation and migration of cervical cancer cells and inhibit apoptosis in vitro, and its mechanism of action may be related to the regulation of the Bax / Bcl-2 apoptosis pathway.

[0124] 2.2 TMEM91 participates in the regulation of autophagy Our preliminary findings using Western blotting experiments suggest that TMEM91 may be involved in the autophagy process. Interference with TMEM91 expression can affect the expression levels of autophagy-related proteins and p62. Figure 1 A). Further research showed that the regulatory effect of TMEM91 on autophagy is dose-dependent: HeLa cells transfected with different doses of GFP-TMEM91 showed that the TMEM91 protein level was correlated with the expression of LC3-II and p62 (A). Figure 2B). To clarify the relationship between TMEM91 and autophagy, we induced autophagy at different time points using a serum starvation method. The results showed that in cells overexpressing TMEM91, the protein expression levels of p62 and LC3-II were lower than those in the control group at all time points. Figure 2 CE); however, in cells with knocked-down TMEM91, the expression of the two showed the opposite trend ( Figure 2 These results suggest that TMEM91 may be involved in regulating the autophagy activity of cervical cancer cells.

[0125] 2.3 TMEM91 interacts with p53 and affects p53 protein expression. The development and progression of cervical cancer are closely related to the dysfunction of the tumor suppressor protein p53. Notably, in over 95% of cervical cancer cases, the p53 gene itself is not mutated; its loss of function is primarily attributed to abnormal protein degradation. Based on this, we found through correlation analysis that the expression level of TMEM91 was significantly negatively correlated with p53. Figure 3 This is consistent with our research hypothesis (A). Further molecular docking simulations showed a stable binding interface between TMEM91 and p53 proteins, suggesting that they may form a direct protein-protein interaction. Figure 3 B). To verify this interaction, we performed an immunoprecipitation experiment in 293T and HeLa cells, which confirmed that TMEM91 and p53 do indeed bind intracellularly. Figure 3 CD). Subsequently, Western blot analysis showed that overexpression of TMEM91 decreased p53 protein levels, while knockdown of TMEM91 increased p53 protein levels. Figure 3 E). To clarify whether TMEM91 regulates p53 at the protein level, rather than affecting its transcription, we detected the mRNA level of p53 using real-time quantitative PCR. The results showed that intervention in TMEM91 expression did not have a significant effect on the mRNA level of p53 (E). Figure 3 (FI). In summary, these data indicate that TMEM91 can negatively regulate p53 at the protein level by binding to and promoting its degradation.

[0126] 2.4TMEM91 degrades p53 via autophagy Given that intracellular protein degradation primarily relies on the ubiquitin-proteasome and autophagy-lysosome pathways, and considering previous experiments suggesting that TMEM91 participates in regulating autophagy, we hypothesized that TMEM91 might mediate p53 degradation through autophagy. To verify this hypothesis, we first treated p53 with the autophagy inhibitor chloroquine (CQ) and the autophagy inducer rapamycin (Rapa). Western blot results showed that CQ treatment significantly inhibited p53 degradation, while Rapa promoted p53 degradation. Figure 4 A). Based on this, we further investigated the role of TMEM91 in this pathway. The results showed that after adding Rapa to cells with knocked-down TMEM91, the degradation of p53 was further intensified compared to the control group. Figure 4 B); Conversely, in cells overexpressing TMEM91, CQ treatment effectively blocked the degradation of p53 (B). Figure 4 C). The above results collectively indicate that TMEM91 likely promotes p53 degradation by activating the autophagy-lysosome pathway.

[0127] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An shRNA that specifically suppresses expression of a TMEM91 gene, characterized in that, comprising the sequence 5'-GCTGCAGTTCTCCAAGGATTC-3' or 5'-GATGTTGAGGACATGTCATCC-3'; said shRNA is prepared by annealing method and used to reduce TMEM91 mRNA and protein level.

2. A recombinant lentiviral vector, characterized in that, comprising the shRNA sequence of claim 1 for stably knocking down TMEM91 expression.

3. A TMEM91 inhibitor, characterized in that, a nucleic acid preparation comprising the shRNA of claim 1.

4. A pharmaceutical composition, characterized by, comprising the TMEM91 inhibitor of claim 3 and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition of claim 4, wherein, further comprising an autophagy modulator, said autophagy modulator is chloroquine or rapamycin.

6. A kit for detecting the interaction of TMEM91 with p53 protein, characterized in that, comprising a TMEM91 specific antibody, a p53 specific antibody and Protein A / G magnetic beads.

7. A method of preparing a TMEM91 knockdown cervical cancer cell model, comprising, comprising the following steps: designing and synthesizing shTMEM91 sequence; constructing recombinant lentivirus vector; transfecting 293T cells to package virus; infecting cervical cancer cell line; puromycin screening stable strains.

8. A method of screening for a TMEM91 inhibitor, characterized in that, comprising: using the cell model of claim 7; detecting cell proliferation by CCK-8 method; detecting p53 protein level by Western Blot.

9. Use of the shRNA of claim 1 in the preparation of a drug for treating cervical cancer.

10. Use of the TMEM91 inhibitor of claim 3 in the preparation of a drug for modulating p53 protein level through autophagy pathway.

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