Application of knocking down TMEM91 to induce renal cancer cell death and tumor immunotherapy for renal cancer

Abstract

The present invention belongs to the field of biomedicine technology, and relates to an application of knocking down TMEM91 to induce renal cancer cell death and tumor immunotherapy for renal cancer. The present invention designs shRNA sequences and siRNA sequences for human and mouse TMEM91, and constructs corresponding shRNA lentiviral silencing vectors. The experimental results confirm that the shRNA of the present invention can significantly silence the expression of TMEM91. In view of TMEM91 as a potential target for the treatment of renal cancer, the shRNA of TMEM91 provided by the present invention and its related biological reagents can be used to study and prepare drugs for the treatment of renal cancer. In addition, the present invention confirms that inhibiting the expression of the TMEM91 gene can inhibit the abnormal proliferation of human renal clear cell carcinoma cells (786-O) and mouse renal cancer cells (RENCA), proving that TMEM91 is a potential new target for the treatment of renal cancer, which is conducive to further studying the occurrence and development mechanism of tumor-related diseases caused by abnormal activation of TMEM91.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to an application of knocking down TMEM91 to induce renal cancer cell death and tumor immunotherapy for renal cancer. Background Art

[0002] RNA interference (RNAi) is a conservative gene defense mechanism, i.e., a sequence-specific post-transcriptional gene silencing (PTGS). With the development of technology in recent years, RNAi technology has important applications in gene function research, disease treatment strategy development, and crop improvement. For example, Inclisiran (Leqvio) developed by Novartis is used to treat hypercholesterolemia. It inhibits the expression of the PCSK9 gene and reduces the level of low-density lipoprotein cholesterol (LDL-C), which is used to reduce the cholesterol content in the blood. In summary, RNAi technology provides new ideas for the treatment of tumors and other diseases.

[0003] Renal carcinoma is a malignant lesion of the kidney caused by various reasons, and there is currently a lack of effective non-invasive treatment methods. Clinical data show that TMEM91 (Transmembrane Protein 91) is significantly highly expressed in renal carcinomas and plays a significant role in promoting cancer in tumors. The expression level of this gene is significantly negatively correlated with the survival rate of patients. TMEM91 is a member of the TMEM family, which generally plays a role in signal transduction, transport and membrane composition in cells. It can be used as a potential target for the treatment of renal carcinomas and is of great significance to drug development and tumor vaccine design.

[0004] Because there is still a lack of inhibitors against TMEM91, it is necessary to develop inhibitors against TMEM91 to improve their specificity and tissue specificity for TMEM91.

[0005] In view of the fact that there is no method for targeted silencing of TMEM91 in the prior art, the present invention aims to provide shRNA sequences, si sequences, primers, applications, shRNA lentiviral vectors and construction methods for targeted silencing of TMEM91, aiming to solve the problem of the current lack of TMEM91 inhibitors and the lack of TMEM91 targeted silencing methods. Summary of the invention

[0006] The present invention first provides an application of knocking down TMEM91 to induce renal cancer cell death and tumor immunotherapy for renal cancer.

[0007] In certain embodiments, the agent comprises shRNA or siRNA.

[0008] In certain embodiments, the sequence of the shRNA includes SEQ ID NO.1-SEQ ID NO.2, SEQ ID NO.17-SEQ ID NO.18.

[0009] In certain embodiments, the sequence of the siRNA includes SEQ ID NO.11-SEQ ID NO.12.

[0010] In certain embodiments, the renal cancer cells are of human origin.

[0011] In certain embodiments, the renal cancer cells are derived from mice.

[0012] The present invention also provides a use of a TMEM91 knockdown agent in inducing renal cancer cell death, wherein the use is for non-therapeutic purposes.

[0013] The present invention also provides a primer for preparing the above reagent, wherein the primer sequence comprises SEQ ID NO.3-SEQ ID NO.6, SEQ ID NO.19-SEQ ID NO.22.

[0014] Finally, the present invention provides a shRNA lentiviral vector, which is used to express the shRNA sequence as described above.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects:

[0016] The present invention designs shRNA sequences and siRNA sequences for human and mouse TMEM91, and constructs corresponding shRNA lentiviral silencing vectors. The experimental results confirm that the shRNA of the present invention can significantly silence the expression of TMEM91. In view of TMEM91 as a potential target for the treatment of renal cancer, the shRNA of TMEM91 provided by the present invention and its related biological reagents can be used to study and prepare drugs for the treatment of renal cancer. In addition, the present invention confirms that inhibiting the expression of the TMEM91 gene can inhibit the abnormal proliferation of human renal clear cell carcinoma cells (786-O) and mouse renal cancer cells (RENCA), proving that TMEM91 is a potential new target for the treatment of renal cancer, which is conducive to further studying the occurrence and development mechanism of tumor-related diseases caused by abnormal activation of TMEM91. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1A is the test result of qPCR;

[0018] Figure 1B Western blot was used to detect protein expression levels;

[0019] Figure 2A The results of PASMC observed under a fluorescence microscope are shown in Figure 2.

[0020] Figure 2B This is a graph showing the detection results of proliferation analysis after counting cells based on the results of fluorescence microscopy observation;

[0021] Figure 3 This is the experimental situation of CCK8;

[0022] Figure 4A The mRNA expression level was detected by qPCR;

[0023] Figure 4B The mRNA expression level was detected by qPCR;

[0024] Figure 5A The staining results for colony counting;

[0025] Figure 5B The number of clones is counted for colony counting;

[0026] Figure 6 The results of qPCR detection of mouse TMEM91;

[0027] Fig. 7A The tumor tissue conditions of mice;

[0028] Figure 7B is the tumor weight of mice;

[0029] Figure 7C The tumor volume of mice;

[0030] Fig.7D For mouse tumor CD8 + Cell infiltration;

[0031] Fig. 7E For mouse tumor CD8 + Cell count status. DETAILED DESCRIPTION

[0032] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0033] Table 1. Experimental materials

[0034]

[0035] Example 1

[0036] Two shRNA sequences targeting silencing human TMEM91

[0037] It contains shRNA designed for TMEM91 gene, and its corresponding target site sequence is: 5'-TTCTCCATGCTGTGTTGTTTC-3' (SEQ ID NO.1);

[0038] with 5'-ACTGCCTCTACAGACCAACAA-3' (SEQ ID NO. 2);

[0039] Two primers for preparing the shRNA sequence as described above, wherein the primer sequences are as follows:

[0040] Upstream primer sequence:

[0041] shTMEM91-1:5'-CCGGTTCTCCATGCTGTGTTGTTTCCTCGAGGAAACAACACAGCATGGAGAATTTTTG-3' (SEQ ID NO. 3);

[0042] ShTMEM91-2:5'-CCGGACTGCCTCTACAGACCAACAACTCGAGTTGTTGGTCTGTAGAGGCAGTTTTTTG-3' (SEQ ID NO.4);

[0043] Downstream primer sequence:

[0044] shTMEM91-1:5'-AATTCAAAAATTCTCCATGCTGTGTTGTTTCCTCGAGGAAACAACACAGCATGGAGAA-3' (SEQ ID NO.5);

[0045] ShTMEM91-2:5'-AATTCAAAAAACTGCCTCTACAGACCAACAACTCGAGTTGTTGGTCTGTAGAGGCAGT-3' (SEQ ID NO. 6);

[0046] Two shRNA lentiviral vectors were designed according to the two target sites, wherein the shRNA lentiviral vectors were used to express the shRNA sequence described above.

[0047] Example 2

[0048] A method for constructing the shRNA lentiviral vector as described above, comprising the following steps:

[0049] The shRNA sequence was synthesized by primer annealing;

[0050] Upstream primer sequence:

[0051] shTMEM91-1:5'-CCGGTTCTCCATGCTGTGTTGTTTCCTCGAGGAAACAACACAGCATGGAGAATTTTTG-3' (SEQ ID NO.3);

[0052] ShTMEM91-2:5'-CCGGACTGCCTCTACAGACCAACAACTCGAGTTGTTGGTCTGTAGAGGCAGTTTTTTG-3' (SEQ ID NO.4);

[0053] Downstream primer sequence:

[0054] shTMEM91-1:5'-AATTCAAAAATTCTCCATGCTGTGTTGTTTCCTCGAGGAAACAACACAGCATGGAGAA-3' (SEQ ID NO.5);

[0055] ShTMEM91-2:5'-AATTCAAAAAACTGCCTCTACAGACCAACAACTCGAGTTGTTGGTCTGTAGAGGCAGT-3' (SEQ ID NO.6):

[0056] The annealing system is shown in Table 2;

[0057] Table 2. shTMEM91 primer annealing system

[0058]

[0059] After preparing the mixed solution, set the PCR instrument program: 95℃--------5min; 94~46℃--decrease 1℃ / min; 46~26℃--decrease 2℃ / min; 4℃---------forever.

[0060] Vector digestion: pLKO.1-puro vector was digested with AgeI and EcoRI at 37°C for 2 hours. The digestion system is shown in Table 3 below;

[0061] Table 3. pLKO.1 vector restriction enzyme digestion system

[0062]

[0063] Carrier recovery:

[0064] (1) Take 1 small bag of TAE buffer (dry powder) and prepare 1000 ml solution (prepared with dd water)

[0065] (2) Prepare 100 ml of agarose gel. Take 1 g of agar and pour it into the flask on the agar gel preparation table. Pour in 100 ml of the TAE solution that has just been prepared. Heat it in a heating box for 60 seconds until it just boils, then let it cool naturally.

[0066] (3) Place the mold and comb on the agar gel preparation table in advance.

[0067] (4) After cooling to room temperature, add 3-5ul of GELRED dye (generally 3-5ul of dye solution is required to prepare 100ml. This dye solution is neurotoxic, so please take precautions)

[0068] (5) After adding the dye, shake well, pour into the mold, and let it cool and solidify naturally. Pull out the comb.

[0069] (6) Prepare 1000 ml of TAE solution and pour it into the electrophoresis tank.

[0070] (7) Place the solidified nucleic acid gel into the electrophoresis tank, add a marker to the first lane, and add the digested vector sample to the remaining lanes.

[0071] (8) Cover the electrophoresis tank and run at 120 V for about 30 min. The bands will be clearly separated.

[0072] (9) Cut the enzyme-cut vector under ultraviolet light and place it into a labeled EP tube.

[0073] (10) Use the Novozyme Gel Recovery Kit (DC301-01) to recover the gel to obtain the double-enzyme-digested vector.

[0074] Connection conversion:

[0075] The annealed primers and the digested vector were configured into a connection system as shown in Table 4.

[0076] Table 4. Ligation of pLKO.1 vector and products

[0077]

[0078] Connect at room temperature for 1 hour, mix with DH-5α competent cells and ice bath for 30 minutes, heat shock at 42℃ for 90 seconds, place on ice for 5 minutes, add anti-antibody LB medium in the bacterial clean bench, and culture on a shaker at 37℃ for 1 hour. Then centrifuge at room temperature, 3000 rpm, 1 minute, remove part of the culture supernatant, keep 500 µL of resuspended precipitate and apply it to ampicillin-resistant LB culture plate, and place it in a 37℃ bacterial incubator for overnight culture. After overnight, pick monoclonal colonies and perform plasmid sequencing to obtain shRNA lentiviral vectors pIKO-shTMEM91-1-Puro and pIKO-shTMEM91-2-Puro for expressing shTMEM91.

[0079] Example 3

[0080] Silencing effect of shTMEM91 lentiviral expression vector on target gene TMEM91

[0081] The pIKO-shTMEM91-1-Puro and pIKO-shTMEM91-2-Puro lentiviral expression vectors prepared in Example 2 were used for lentiviral packaging, infected into 786-O cell lines, and the silencing effect of the target gene RhoB was verified by Western Blot, specifically comprising the following steps:

[0082] 1. Lentiviral packaging of shTMEM91 lentiviral interference vector:

[0083] 1.1 Use DMEM medium (10% fetal bovine serum) to culture 293T cells. One day before transfection, inoculate 293T cells in a 100 mm plate and culture for 12 to 24 hours.

[0084] 1.2 When the cell density reaches 70-80%, the plasmid mixture required for each group of the transfection system is 2μg VSVG; 3μg PAX2 packaging plasmid and 5μg pIKO-shTMEM91-1-Puro or pIKO-shTMEM91-2-Puro shRNA lentiviral recombinant plasmid. After the above plasmids are fully mixed, add 500μl jetprime buffer and 24uljetprime. The experiment uses the shcontrol lentiviral vector targeting non-target gene sequences as a control.

[0085] 1.3 Mix the transfection reagent and plasmid mixture thoroughly and let stand at room temperature for 10 minutes. Slowly add the mixture drop by drop into a 100mm culture dish and culture it in a 37℃ 5% CO2 incubator. After 4 hours, replace with fresh DMEM medium (10% fetal bovine serum). After another 48 hours of culture, collect the supernatant. Centrifuge at 1500rpm for 10 minutes, filter the collected supernatant with a 0.45µm filter to obtain the corresponding virus solution. Add 5×Lentivirus Concentration Reagent (genstar C103-05) to the virus solution at a ratio of 4:1, mix and store at 4℃ overnight. After overnight, centrifuge at 12000rpm for 15 minutes, pour off the supernatant, resuspend with 1ml PBS, and store at -80℃.

[0086] 2.786-O Cell Culture

[0087] Cell culture: 786-O (human renal clear cell carcinoma cells) were cultured in 1640 complete medium (Pnosel) containing 10% FBS. Cells with good growth status were collected, centrifuged and counted, and 6×10 5 Each well was inoculated into a 60 mm dish and cultured at 37°C, 5% CO2 for 24 h.

[0088] 3. shTMEM91 lentivirus infection 786-O:

[0089] 3.1 786-O cells were seeded in 6-well plates at a cell density of 1.5×10 5 cells / well.

[0090] 3.2 After 12-24 hours of culture, 786-O was infected with a mixture of 2 ml 1640 + 100 μL shTMEM91-1 or shTMEM91-2 lentivirus solution + 2.1 μL polybrene (polybrene, final concentration of 10 μg / μL). 786-O was infected with shcontrol lentiviral expression vector and lentivirus targeting non-target gene sequences as a control for shTMEM91 lentiviral expression vector and lentivirus.

[0091] 3.3 Replace the culture medium with fresh one 24 hours after virus infection.

[0092] 3.4 After the cells were infected with the virus for 48 h, puromycin was added to the culture medium at a final concentration of 2 μg / μL and the cells were cultured until a cell line stably infected with the shTMEM91-1 or shTMEM91-2 lentivirus was selected.

[0093] 4. qPCR detection of mRNA expression levels

[0094] 4.1 Lyse 786-O cells stably infected with shTMEM91-1, shTMEM91-2, and shcontrol lentiviruses and extract RNA using an RNA extraction kit. After extraction, use a nucleic acid quantifier to measure RNA content

[0095] 4.2 Use the extracted RNA and reverse transcription kit for reverse transcription. The configuration system is shown in Table 5 below.

[0096] Table 5. Reaction system

[0097]

[0098] 4.3 qPCR primer design

[0099] Design of qPCR primers targeting TMEM91:

[0100] Upstream primer sequence: 5′-AGGGGTTTGCAGTTCCTGTC-3′ (SEQ ID NO. 7);

[0101] Downstream primer sequence: 5′-GTGGTCGTGGGGTAGAAATGG-3′ (SEQ ID NO. 8);

[0102] Design of qPCR primers targeting GAPDH

[0103] Upstream primer sequence: 5′-agaaggctggggctcatttg-3′ (SEQ ID NO. 9);

[0104] Downstream primer sequence: 5′-aggggccatccacagtcttc-3′ (SEQ ID NO. 10);

[0105] 4.4 qPCR system configuration

[0106] Table 6. Reaction system

[0107]

[0108] Three replicate wells were set up for each sample and added to a 96-well plate, for a total of 18 wells.

[0109] 4.5 On-machine testing, the setting procedure is as follows

[0110] Table 7. Reaction Procedure

[0111]

[0112] Denaturation and annealing / extension were set for 40 cycles, and the program was terminated at 95°C for 5s and 65°C for 5s. The mRNA levels of TMEM91 in each sample were obtained. Figure 1A .

[0113] 5. Western blot to detect protein expression level:

[0114] 5.1 Lyse 786-O cells stably infected with shTMEM91-1, shTMEM91-2, and shcontrol lentiviruses, collect protein samples, and after protein quantification, take 20 μg of protein sample for SDS-PAGE electrophoresis at a constant voltage of 120 V.

[0115] 5.2 Transfer: 25V, 1.3A, 10min. After transfer, place the membrane in 5% skimmed milk powder blocking solution for 1h, and then wash the membrane twice with TBST.

[0116] 5.3 Add TMEM91 primary antibody (1:1000 dilution) in antibody diluent and place on a shaker at 4°C overnight.

[0117] 5.4 On the second day, wash the membrane three times with TBST, each time for 5 minutes, add the secondary antibody prepared with antibody diluent (1:5000 dilution), incubate at room temperature for 1 hour and then develop the color.

[0118] The results are shown in Figure 1B. Compared with the control group infected with the shcontrol lentivirus, the TMEM expression levels in 786-O infected with the shTMEM91-1 and shTMEM91-2 lentiviruses were significantly downregulated, indicating that shTMEM91-1, shTMEM91-2 and their lentiviral expression vectors prepared using the shRNA primers designed by the present invention have a significant silencing effect on TMEM91 in 786-O.

[0119] 6.shTMEM91 can inhibit the proliferation of 786-O

[0120] 6.1 Edu cell proliferation experiment: To explore the regulatory effect of down-regulating TMEM91 expression on 786-O, the aforementioned shcontrol, shTMEM91-1, and shTMEM91-2 lentiviral expression vectors were transfected at 37°C and 5% CO2, and then the cell proliferation level was determined by EdU cell proliferation assay. EdU cell proliferation assay kit (Ribo Bio) was used for EdU labeling, and the experimental operation was performed according to the kit instructions. In brief, about 5×10 4The cells were plated in a 24-well plate and cultured for 24 hours before the transfection experiment was performed. Then 20 μM EdU was added and cultured for another 5 hours. After the cells were taken out, they were fixed with 4% paraformaldehyde at room temperature for 30 minutes, permeabilized with 0.5% Triton X-100 for 10 minutes, washed with PBS, and then 150 μL 1× Apollo staining reaction solution was added to each well of cells for 30 minutes. DNA was stained with 1× Hochest 33342 (150 μL per well) for 5 minutes, and photographed under a fluorescence microscope. The results showed that compared with the shcontrol group, the proliferation of 786-O was reduced by 80% after transfection with shTMEM91-1 and shTMEM91-2 lentiviral expression vectors, such as Figure 2A As shown in Figure 2B, Figure 2A The results of PASMC observed under a fluorescence microscope. Figure 2B The experimental results show that the expression level of TMEM91 in 786-O cells can be significantly down-regulated by using shTMEM91-1, shTMEM91-2 and lentiviral expression vectors designed by the present invention, and the abnormal proliferation of 786-O cells can be inhibited.

[0121] 6.2 CCK8 experiment: To explore the regulatory effect of down-regulating TMEM91 expression on 786-O, the aforementioned shcontrol, shTMEM91-1, and shTMEM91-2 lentiviral expression vectors were transfected at 37°C 5% CO2, and then the cell proliferation level was determined by the CCK8 method. 100 μL of 2000 cells were added to each well. After 12 hours of culture, 10 μL of CCK-8 solution was added to each well. Incubate in the cell culture incubator for another 2 hours. Measure the absorbance at 450 nm. At this time, mark 0h, and measure it at 24h, 48h, and 72h, respectively, see Figure 3 .

[0122] Example 4

[0123] Two siRNA sequences targeting silencing human TMEM91

[0124] Contains siRNA designed for TMEM91 gene, and its corresponding target site sequence:

[0125] 5'-GCCCUAGUCUUCGUGAGCUTT-3' (SEQ ID NO. 11);

[0126] with 5'-GGCAGCCGUCUUUCACCAUTT-3' (SEQ ID NO. 12);

[0127] Sense sequence:

[0128] siTMEM91-1:5'-GCCCUAGUCUUCGUGAGCUTT-3' (SEQ ID NO. 13);

[0129] siTMEM91-2:5'-GGCAGCCGUCUUUCACCAUTT-3' (SEQ ID NO. 14);

[0130] Antisense sequence:

[0131] siTMEM91-1:5'-AGCUCACGAAGACUAGGGCTT-3' (SEQ ID NO. 15);

[0132] siTMEM91-2:5'-AUGGUGAAAGACGGCUGCCTT-3' (SEQ ID NO. 16);

[0133] Two siRNAs and one sicontrol were purchased from Jima Biotechnology;

[0134] 1. siRNA transfection cells:

[0135] 1.1 786-O cells were cultured as described above and 786-O cells were seeded in 6-well plates at a cell density of 1.5×10 5 cells / well.

[0136] 1.2 After 12 days of culture, 786-O cells were transfected with 2 ml of 1640 + 5 μL siTMEM91-1 or siTMEM91-2 siRNA + 5 μL polybrene + 200 μL polybrene Buffer. 786-O cells were transfected with shcontrol siRNA targeting non-target gene sequences as a control for siTMEM91 gene.

[0137] 1.3 After 6 hours of transfection, fresh culture medium was replaced to construct siTMEM91-1 and siTMEM91-2 si cell lines.

[0138] 2. qPCR detection of mRNA expression levels

[0139] Follow the above method, see Figure 4A .

[0140] 3. Western blot to detect protein expression levels

[0141] Follow the above method, see Figure 4B .

[0142] 4. Plate cloning experiment:

[0143] 4.1 According to the above transfection method, 24 hours after transfection, 786-O was seeded in a 6-well plate with a cell density of 700 cells / well, and 3 parallel samples were set up.

[0144] 4.2 Place the cells in a cell culture incubator at 37°C, 5% CO2 and saturated humidity for 14 days. Change the medium every 3 days, and be careful to add the culture medium slowly to avoid blowing the cells.

[0145] 4.3 When colonies are visible, stop the culture, wash with PBS, and then fix the cells with paraformaldehyde for 15-30 minutes.

[0146] 4.4 Stain the cells with crystal violet for 30 minutes.

[0147] 4.5 Slowly wash away the staining solution with running water, and observe and take pictures after air drying.

[0148] 4.6 Use ImageJ software to count colonies. Figure 5A and Figure 5B .

[0149] Example 5

[0150] Two shRNA sequences targeting silencing mouse TMEM91

[0151] Contains shRNA designed for TMEM91 gene, and its corresponding target site sequence: 5'-CTGACTTTGAGGACACATTAT-3' (SEQ ID NO. 17);

[0152] with 5'-ACCCTTACTGCCAAGCATAAC-3' (SEQ ID NO. 18);

[0153] Two primers for preparing the shRNA sequence as described above, wherein the primer sequences are as follows:

[0154] Upstream primer sequence:

[0155] shTMEM91-M-1:5'-CCGGCTGACTTTGAGGACACATTATCTCGAGATAATGTGTCCTCAAAGTCAGTTTTTG-3' (SEQ ID NO. 19);

[0156] ShTMEM91-M-2:5'-CCGGACCCTTACTGCCAAGCATAACCTCGAGGTTATGCTTGGCAGTAAGGGTTTTTTG-3' (SEQ ID NO. 20);

[0157] Downstream primer sequence:

[0158] shTMEM91-M-1:5'-AATTCAAAAACTGACTTTGAGGACACATTATCTCGAGATAATGTGTCCTCAAAGTCAG-3' (SEQ ID NO. 21);

[0159] ShTMEM91-M-2:5'-AATTCAAAAAACCCTTACTGCCAAGCATAACCTCGAGGTTATGCTTGGCAGTAAGGG-3' (SEQ ID NO. 22);

[0160] 1. Construction of vector and lentivirus

[0161] The aforementioned plasmid and lentiviral construction methods were used.

[0162] 2. Cultivation of RENCA cells

[0163] Cell culture: RENCA (rat renal carcinoma cells) were cultured in RENCA complete medium (Prunosai) containing 10% FBS. Cells with good growth status were collected, centrifuged and counted, and 6×10 5 Each well was inoculated into a 60 mm dish and cultured at 37°C, 5% CO 2 for 24 h.

[0164] 3. shTMEM91 lentivirus infection RENCA:

[0165] 3.1 RENCA was seeded in 6-well plates at a cell density of 1.5×105 cells / well.

[0166] 3.2 After 12-24 hours of culture, RENCA cells were infected with a mixture of 2 ml RENCA medium + 100 μL shTMEM91-M-1 or shTMEM91-M-2 lentivirus solution + 2.1 μL polybrene (polybrene, final concentration of 10 μg / μL). RENCA cells were infected with shcontrol lentiviral expression vectors and lentivirus targeting non-target gene sequences as controls for shTMEM91 lentiviral expression vectors and lentivirus.

[0167] 3.3 Replace the culture medium with fresh one 24 hours after virus infection.

[0168] 3.4 After the cells were infected with the virus for 48 h, puromycin was added to the culture medium at a final concentration of 1 μg / μL and the cells were cultured continuously until a cell line stably infected with the shTMEM91-M-1 or shTMEM91-M-2 lentivirus was selected.

[0169] 4. qPCR detection of mRNA expression levels

[0170] qPCR primer design

[0171] Design of qPCR primers for mouse TMEM91:

[0172] Upstream primer sequence: 5′-CACTGAAACTGCGTCTCTGGA-3′ (SEQ ID NO. 23);

[0173] Downstream primer sequence: 5′-CAAGGCTCAGGTCAGGAAAGT-3′ (SEQ ID NO. 24);

[0174] The remaining steps are the same as before: the results are shown in Figure 6 .

[0175] Example 6

[0176] Mouse subcutaneous tumor formation experiment

[0177] To investigate the regulatory effect of down-regulating TMEM91 expression on tumorigenesis in mice

[0178] 6.1 Culture the selected RENCA cells in RENCA medium in a cell culture incubator at 37°C with 5% CO2.

[0179] 6.2 Digest the cells that have grown to 80% to 90% of the culture dish with trypsin, centrifuge at 1800 rpm, remove the supernatant, add PBS to wash the cells, resuspend them in PBS again, count the cells, and adjust the number to 1×106 cells / ml.

[0180] 6.3 Prepare enough cell suspension according to the number of Balb / c mice for inoculation. Store the prepared cell suspension in a 4℃ refrigerator and inoculate mice as soon as possible.

[0181] 6.4 The injection site was uniformly 0.3 cm from the axilla of the right forelimb of the mouse. After disinfection with iodine cotton balls, a 1 ml syringe was used to extract the mixed cell suspension (density 1×10 6 / ml) 0.1ml, after the syringe needle pierces the mouse skin, slightly lift the needle, move to the injection site, and slowly inject the cell suspension. After the injection is completed, press the needle insertion site with a sterile cotton ball, and dispose of the experimental equipment and waste harmlessly. Put the newly modeled mice into a cage with new bedding, and observe the mental state, activity and eating of the nude mice the next day, and whether there is redness or swelling at the injection site.

[0182] 6.5 Tumor measurement: After the tumor is visible to the naked eye, the short diameter (W) and long diameter (L) of the transplanted tumor are measured with a vernier caliper. The formula V (cm 3 )=L×W2 / 2 Calculate the tumor volume and measure the tumor size twice a week.

[0183] 6.6 Final sampling: 23 days after the mice were implanted with tumors, tumor tissues were collected and the volume and weight of the tumors were calculated. Fig. 7A , Figure 7B and Figure 7C .

[0184] Example 7

[0185] Immunohistochemistry CD8 + Staining experiments

[0186] To investigate the regulatory effect of down-regulating TMEM91 expression on tumorigenic immune cell infiltration in mice

[0187] 7.1 Immerse the tumor tissue in the mouse in formalin tissue fixative for 48 hours.

[0188] 7.2 Use an automatic dehydrator to dehydrate, transparentize, and wax-impregnate the tissue.

[0189] 7.3 Use an embedding machine to remove the wax-soaked tissue, place it in a mold, and embed it in paraffin.

[0190] 7.4 Use a microtome to evenly cut the embedded wax block into 5 μm thick slices and place them on a glass slide.

[0191] 7.5 Use xylene and different concentrations of ethanol for dewaxing, and then rinse with PBS buffer.

[0192] 7.6 Place the slide in citrate buffer and heat until boiling. Stop heating for 5 minutes and continue heating until boiling. Repeat twice and then slowly cool to room temperature.

[0193] 7.7 After the sections were thoroughly washed with PBS buffer, they were treated with 3% hydrogen peroxide and incubated at 37°C for 40 minutes to block endogenous peroxidase activity.

[0194] 7.8 After washing the sections thoroughly with PBS buffer, block with normal serum or protein (such as bovine serum albumin) and incubate at 37°C for 1 hour to block non-specific binding sites.

[0195] 7.9 Add CD8 primary antibody to the sections and incubate overnight at 4°C.

[0196] 7.10 After washing the sections thoroughly with PBS buffer, incubate with secondary antibody that matches the species of the primary antibody for 1 hour.

[0197] 7.11 After washing the sections thoroughly with PBS buffer, use a substrate such as DAB (3,3'-diaminobenzidine) to develop the color and produce a visible color reaction.

[0198] 7.12 Stain the nuclei using a stain such as hematoxylin to better localize the proteins.

[0199] 7.13 Use ethanol and xylene for dehydration and transparency, and use resin or other mounting media to seal the sections for long-term storage and observation.

[0200] 7.14 Observe the sections using a microscope and analyze protein expression and localization. Fig.7D and Fig. 7E .

[0201] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a TMEM91 knockdown reagent in the preparation of a drug for inducing renal cancer cell death and tumor immunotherapy for renal cancer, characterized in that: The reagent comprises shRNA or siRNA, and the sequence of the shRNA comprises SEQ ID NO.1-SEQ ID NO.2, SEQ ID NO.17-SEQ ID NO.18; The sequence of the siRNA includes SEQ ID NO.11-SEQ ID NO.

12.

2. The use according to claim 1, characterized in that: The renal cancer cells are derived from humans.

3. The use according to claim 1, characterized in that: The renal cancer cells were derived from mice.

4. A shRNA lentiviral vector, characterized in that: The shRNA lentiviral vector is used to express the shRNA sequence according to claim 1.

Images