Use of tpm3p9 protein and rbm4 protein in kidney cancer

Abstract

The application discloses application of TPM3P9 protein and RBM4 protein in kidney cancer. The application finds that high expression of RBM4 indicates better prognosis of a kidney cancer patient, low expression of TPM3P9 and high expression of RBM4 are the best for the prognosis of a patient, therefore, the RBM4 protein or the RBM4 protein and the TPM3P9 protein can be used together as a kidney cancer clinical diagnosis or prognosis biomarker. The application also finds through experiments that the proliferation and growth of kidney cancer cells can be regulated by regulating the expression amount of the TPM3P9 protein, therefore, the RBM4 protein and the TPM3P9 protein can be used together for developing a medicine for treating kidney cancer or proliferation and growth of kidney cancer cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to application of TPM3P9 protein and RBM4 protein in kidney cancer. BACKGROUND

[0002] Kidney cancer is a common malignant tumor of the urinary system, and its incidence and mortality rate have shown a rising trend in recent years, seriously endangering human life safety. Due to the non-obvious early symptoms of kidney cancer, most kidney cancer patients have developed to the middle and late stages at the time of clinical diagnosis, missing the best treatment opportunity. Local kidney cancer can be treated by radical nephrectomy and cryoablation. Although the method of kidney resection has a significant effect on the treatment of local kidney cancer, more than 15% of kidney cancer patients will develop distant metastasis, which requires systemic treatment and is accompanied by a high mortality rate, and the 5-year survival rate of metastatic kidney cancer patients is less than 10%. Kidney cancer is not sensitive to traditional chemotherapy and radiotherapy, and the current drug treatment for kidney cancer is mainly based on the targeting therapy of vascular endothelial growth factor (VEGF) and mTOR pathway, but the treatment effect is still not ideal. Therefore, it is imperative to further explore the biomarkers and therapeutic targets of kidney cancer and develop new targeted drugs based on this.

[0003] With the rapid development of sequencing technology and proteomics technology, more and more research reports show that part of the non-coding genes accounting for about 98% of the human genome can encode new proteins, and these new proteins are usually translated from small open reading frames (sORF) on lncRNA and play an important regulatory role in tumor biology. Mass spectrometry technology is considered to be the most direct tool for identifying new proteins, but mass spectrometry technology relies on a reference database established by protein sequences. Transcriptomics (RNC-seq and Ribo-seq) can discover a large number of RNAs with translation function in cells, which makes up for the lack of new protein database in mass spectrometry technology. Therefore, the combined use of proteomics and transcriptomics technology will help to discover and identify new proteins.

[0004] RBM4 protein is a tumor suppressor protein that can inhibit the function by regulating the alternative splicing of target gene pre-mRNA (pre-mRNA). TPM3P9 (Homo sapiens tropomyosin 3 pseudogene 9) is a long non-coding RNA. The function of TPM3P9 and RBM4 protein in the human body and its relationship with kidney cancer have not been reported yet. SUMMARY

[0005] The primary purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide the use of a reagent for detecting the expression level of RBM4 protein in the preparation of a product for diagnosing or assisting in the diagnosis of kidney cancer, and / or a product for predicting the prognosis risk of kidney cancer.

[0006] Another object of the present application is to provide the use of the reagent for detecting the expression level of the TPM3P9 protein and the RBM4 protein in the preparation of a product for diagnosing or assisting in the diagnosis of kidney cancer, and / or a product for predicting the prognosis risk of kidney cancer.

[0007] Still another object of the present application is to provide the use of the TPM3P9 protein and the RBM4 protein in combination in the preparation of a medicine for treating kidney cancer and / or inhibiting the proliferation and growth of kidney cancer cells.

[0008] The object of the present application is achieved by the following technical solutions:

[0009] The use of the reagent for detecting the expression level of the RBM4 protein in the preparation of a product for diagnosing or assisting in the diagnosis of kidney cancer, and / or a product for predicting the prognosis risk of kidney cancer.

[0010] The accession number of the RBM4 protein sequence in the Uniprot database (protein database) is: Q9BWF3 (https: / / www.uniprot.org / uniprotkb / Q9BWF3 / entry).

[0011] The use of the reagent for detecting the expression level of the TPM3P9 protein and the RBM4 protein in the preparation of a product for diagnosing or assisting in the diagnosis of kidney cancer, and / or a product for predicting the prognosis risk of kidney cancer.

[0012] The amino acid sequence of the TPM3P9 protein is shown as SEQ ID NO. 1.

[0013] The accession number of the RBM4 protein sequence in the Uniprot database (protein database) is: Q9BWF3 (https: / / www.uniprot.org / uniprotkb / Q9BWF3 / entry).

[0014] The coding gene sequence of the TPM3P9 protein is any one of the following sequences:

[0015] (a) The accession number in NCBI is NR_003148.3;

[0016] (b) as shown in SEQ ID NO. 2.

[0017] The reagent for detecting the expression level of the TPM3P9 protein and the RBM4 protein can be a reagent for detecting the TPM3P9 protein and the RBM4 protein respectively, or a reagent for detecting the TPM3P9 protein and the RBM4 protein simultaneously.

[0018] The product includes a kit, a test paper or a chip.

[0019] Use of TPM3P9 protein (as a target) and RBM4 protein in combination in the preparation of a medicament for treating kidney cancer and / or inhibiting the proliferative growth of kidney cancer cells.

[0020] The treatment of kidney cancer or inhibition of the proliferative growth of kidney cancer cells is achieved by knocking down the TPM3P9 protein and overexpressing the RBM4 protein; knocking down the TPM3P9 protein can inhibit the proliferative and growth ability of kidney cancer cells (inhibit the clonogenicity of kidney cancer cells), and overexpressing the RBM4 protein can also inhibit the proliferative and growth ability of kidney cancer cells.

[0021] The inoculation mode of the medicament is preferably subcutaneous injection or intravenous injection.

[0022] The kidney cancer includes renal clear cell carcinoma, etc.

[0023] The medicament for inhibiting the proliferative growth of kidney cancer cells includes a medicament for regulating the alternative splicing function of kidney cancer cells. Silencing the TPM3P9 protein can enhance the alternative splicing function of kidney cancer cells, and interfering with the RBM4 protein inhibits the alternative splicing function, that is, the TPM3P9 regulates the alternative splicing function of kidney cancer by interacting with the RBM4, thereby regulating the proliferative growth of kidney cancer.

[0024] The knocking down of the TPM3P9 protein is achieved by silencing or knocking out the TPM3P9 gene (knocking down the TPM3P9 protein); for example, by small interfering RNA (siRNA) silencing the TPM3P9 gene, or by sgRNA targeted knockout of the TPM3P9 gene, etc.; wherein,

[0025] The siRNA sequence for silencing the TPM3P9 gene is preferably as follows:

[0026] si-TPM3P9-sense: 5'-GGAAGAGAUGAAGAUUCUUTT-3' (SEQ ID NO. 5);

[0027] si-TPM3P9-antisense: 5'-AAGAAUCUUCAUCUCUUCCTT-3' (SEQ ID NO. 6);

[0028] The sgRNA sequence for targeted knockout of the TPM3P9 gene is preferably any one of the following sequences:

[0029] sgRNA#1: 5'-CACCGCATTCAGGTCAAGCAAAGTCGTTT-3' (SEQ ID NO. 7);

[0030] sgRNA#2: 5'-CACCGGCAGAGACCCATGCTGAGTGTTT-3' (SEQ ID NO. 8).

[0031] The overexpression of the RBM4 protein is preferably achieved by the following method:

[0032] (1) The coding gene sequence of the RBM4 protein and the pLVX-Puro Vector plasmid are digested by restriction enzymes EcoR I and BamH I, and then connected by T4 ligase to obtain a recombinant plasmid pLVX-Puro-RBM4.

[0033] (2) The recombinant plasmid pLVX-Puro-RBM4 is transfected into 293T cells together with a packaging plasmid, and then the supernatant of the transfected cells is taken out, centrifuged and filtered to remove cell debris, and then the supernatant is used to infect kidney cancer cells, and after resistance screening, a cell strain overexpressing the RBM4 protein is obtained.

[0034] The resistance screening in step (2) is screening by 2 μg / mL puromycin.

[0035] The kidney cancer cells in step (2) are human kidney cancer cells; preferably human kidney cancer cells ACHN and / or human renal clear cell adenocarcinoma cells 786-O.

[0036] The present application combines cell proliferation phenotype and mouse xenograft experiments in vitro to find that high expression of the new protein TPM3P9 promotes the proliferation of kidney cancer cells, while high expression of the RBM4 protein inhibits the proliferation of kidney cancer cells. Mechanically, the cancer-promoting protein TPM3P9 interacts with the splicing factor RBM4 and regulates alternative splicing to regulate the growth and proliferation of kidney cancer cells. Therefore, targeting TPM3P9 and RBM4 proteins in clinical practice can inhibit the proliferation of kidney cancer cells.

[0037] The present application has the following advantages and effects relative to the prior art:

[0038] 1. The present application first finds that the TPM3P9 protein encoded by lncRNA is related to the proliferation of kidney cancer. The TPM3P9 protein, as a cancer-promoting protein, can effectively promote the growth and proliferation of kidney cancer cells. It interacts with the splicing factor RBM4, regulates the alternative splicing of the TCF7L2 gene, activates the NF-κB pathway, and significantly promotes the proliferation of kidney cancer. Therefore, TPM3P9 and RBM4 can be used as a regulatory target for cancer cell proliferation.

[0039] 2、The application finds that the tumor proliferation and growth can be regulated by regulating the expression amount of TPM3P9 protein. In renal cancer, the cancer-promoting protein TPM3P9 interacts with the splicing factor RBM4, regulates alternative splicing and further regulates the growth and proliferation of renal cancer cells, so that targeting TPM3P9 and RBM4 proteins can inhibit the proliferation and growth of renal cancer cells, that is, TPM3P9 and RBM4 can be used as drug targets for clinical diagnosis of renal cancer, and can be used for developing drugs for treating renal cancer or proliferation and growth of renal cancer cells.

[0040] 3、The application finds that high expression of RBM4 indicates better prognosis of renal cancer patients, so that the RBM4 protein can be used as an effective biomarker for clinical diagnosis of renal cancer. At the same time, the application also finds that the prognosis of patients in the group with high expression of TPM3P9 and low expression of RBM4 is the worst, and the prognosis of patients with low expression of TPM3P9 and high expression of RBM4 is the best, so that TPM3P9 and RBM4 can be used as biomarkers for clinical diagnosis or prognosis of renal cancer. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 is an expression diagram of a new protein TPM3P9; wherein, A is a table showing TPM3P9 (TPM3P9 is located on chromosome 19 and consists of 2 exons, and the encoded protein is 92 amino acids); B is a specific peptide segment of TPM3P9 identified by mass spectrometry.

[0042] Figure 2 is a graph of experimental results of the role of RBM4 in the prognosis of renal cancer; wherein, A is that the expression of RBM4 in renal cancer is lower than that in the adjacent cancer tissue; B is that the expression of RBM4 in high-stage renal cancer samples is reduced; C is that the expression of RBM4 in metastatic renal cancer is lower than that in non-metastatic renal cancer; D is that high expression of RBM4 is related to good prognosis of renal cancer.

[0043] Figure 3 is a curve graph of the relationship between the expression amount of RBM4 protein and the survival time of renal cancer patients; wherein, A is overall survival curve (OS); B is disease-free survival curve (DFS).

[0044] Figure 4 is a curve graph of the relationship between the expression amount of TPM3P9 and RBM4 proteins and the survival time of renal cancer patients; wherein, A is the immunohistochemical staining of TPM3P9 and RBM4 proteins in 4 renal cancer patients; B is overall survival curve (OS); C is disease-free survival curve (DFS).

[0045] Figure 5Figure is the result of western blotting experiment of the interaction between TPM3P9 and RBM4; wherein, A is that TPM3P9 immunoprecipitates RBM4; B is that RBM4 immunoprecipitates TPM3P9; C is that overexpression of TPM3P9 protein does not affect the protein expression of RBM4 in ACHN and 786-O cells.

[0046] Figure 6 Figure is the effect of TPM3P9 and RBM4 on the proliferation of renal cancer cells; wherein, A is the effect of TPM3P9 and RBM4 on the proliferation of ACHN and 786-O cells (CCK-8 experiment); B, C and D are the effect of TPM3P9 and RBM4 on the growth of renal cancer cells in mice; E is the change of body weight of mice during the in vivo experiment.

[0047] Figure 7 Figure is the expression of TPM3P9 in renal cancer cell lines ACHN and 786-O when overexpressed and knocked down.

[0048] Figure 8 Figure is the effect of TPM3P9 protein on the proliferation and growth of renal cancer cells in vitro; wherein, A is that TPM3P9 protein is successfully knocked down in renal cancer cells; B is that silencing TPM3P9 protein inhibits the growth ability of renal cancer cells (CCK-8 experiment); C is that knocking down TPM3P9 protein inhibits the colony formation ability of renal cancer cells; D is that knocking down TPM3P9 protein inhibits the proliferation of renal cancer cells (EdU experiment).

[0049] Figure 9 Figure is the effect of TPM3P9 protein on the proliferation and growth of renal cancer cells in mice; wherein, A is that knocking down TPM3P9 protein inhibits the growth curve of renal cancer cells in mice; B is that knocking down TPM3P9 protein inhibits the growth of renal cancer cells in mice; C is that knocking down TPM3P9 protein inhibits the weight of subcutaneous tumor in mice; D is the change of body weight of mice during the in vivo experiment. DETAILED DESCRIPTION

[0050] The application will be described in further detail below with reference to the examples. The reagents, methods and equipment used in the application are conventional in the art unless specifically stated otherwise. The test methods in the following examples, unless otherwise specified, are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the application are commercially available.

[0051] Example 1 Identification of new protein TPM3P9

[0052] 1. The present application finds that TPM3P9 (Homo sapiens tropomyosin 3 pseudogene 9, long non-coding RNA) (NCBI Accession No.: NR_003148.3, 2932 bp) can encode a new protein, and the coding length is 92 amino acids, which is named as TPM3P9 protein; wherein,

[0053] The sequence of TPM3P9 protein (SEQ ID NO. 1) is as follows:

[0054] MDEQIRLMDQNLKCLSAAEEKYSQKEDKCEEEMKILTDNLKEAETHAELAERSVAKL EKTIDDLEDKLKCTKEEHLCTQRMLDQTLLDLNEM;

[0055] The sequence of TPM3P9 gene (SEQ ID NO. 2) is as follows:

[0056] ATGGATGAGCAGATCAGACTGATGGACCAGAACCTGAAGTGTCTGAGTGCAGCTGAAGAAAAGTACTCTCAAAAAGAAGACAAATGTGAGGAAGAGATGAAGATTCTTACTGATAATCTCAAGGAGGCAGAGACCCATGCTGAGTTGGCTGAGAGATCAGTAGCCAAGCTGGAAAAGACAATTGATGACTTGGAAGATAAACTGAAATGCACCAAAGAGGAACACCTCTGTACACAAAGGATGCTGGACCAGACTTTGCTTGACCTGAATGAGATGTAG.

[0057] The coding sequence of TPM3P9 is as shown in Figure 1 A, and the non-coding RNA-TPM3P9 encodes a protein with a size of 10 kDa.

[0058] 2. The present application further provides protein evidence for the identification of TPM3P9 by mass spectrometry, and the specific experimental steps are as follows:

[0059] (1) Protein digestion: the protein solution is obtained by lysing the renal cancer cell lines ACHN and 786-O cells (purchased from ATCC), and then 200 μg of protein is subjected to enzymolysis. After reduction and alkylation treatment, mass spectrometry grade trypsin (Beijing Shengxia Protein) is added at a mass ratio of trypsin: protein of 1:30-40, and the mixture is placed at 37℃ for 16 hours of enzymolysis. Finally, the peptides obtained by enzymolysis are freeze-dried by a vacuum drying machine.

[0060] (2) Desalting: The freeze-dried enzymatic peptides were re-dissolved with mass spectrometry grade water (Thermo Fisher Scientific) containing 0.5% (v / v) TFA (trifluoroacetic acid), and after detecting the concentration, 15 μg of the enzymatic peptides were desalted by a C18 column (Thermo Fisher Scientific). Finally, the desalted peptides were freeze-dried by a vacuum dryer.

[0061] (3) Mass spectrometry identification: The desalted peptides were re-dissolved with mass spectrometry grade water containing 0.1% (v / v) FA (formic acid), and after detecting the concentration, they were prepared into a peptide solution with a concentration of 0.5 μg / μL, and iRT reagent (Shanghai Yisuan Biological) was added. Finally, 6 μL of the solution was added to the mass spectrometry sample bottle, and an Orbitrap Fusion Lumos three-in-one mass spectrometer (Thermo Fisher Scientific) was used for mass spectrometry identification. The specific peptides of TPM3P9 identified by mass spectrometry are shown in Figure 1 B.

[0062] Example 2 Prognostic value of RBM4 in renal cancer

[0063] 1. The renal cancer and paracancer data in the TCGA (The Cancer Genome Atlas, https: / / portal.gdc.cancer.gov / ) dataset were analyzed. The results are shown in Figure 2 It was found that the expression of RBM4 in renal cancer tissue was significantly lower than that in its paracancer tissue ( Figure 2 A), and RBM4 was expressed lower in higher T stage of renal cancer patients (divided into T1 stage (I), T2 stage (II), T3 stage (III), and T4 stage (IV)) Figure 2 B), RBM4 was expressed lower in metastatic renal cancer patients (metastatic patients (N1), non-metastatic patients (N0)) Figure 2 C), high expression of RBM4 contributed to good prognosis of renal cancer Figure 2 D), thus indicating that RBM4 played a tumor inhibiting function in renal cancer.

[0064] 2. The tissue chip of 369 clinical samples of renal cancer patients (the tissue chip was from the Center for Cancer Prevention and Treatment of Sun Yat-sen University) was selected for RBM4 immunohistochemical detection. The patients were divided into two groups, RBM4 high expression group and low expression group, according to the RBM4 staining score, and the survival curve was fitted according to the corresponding survival time. The specific steps are as follows:

[0065] (1) Immunohistochemical detection: The above tissue sample chip and tissue sample were respectively incubated with anti-RBM4 polyclonal antibody (Wuhan Proteintech Group Co., Ltd.) at 4°C overnight according to 1:500 (volume ratio), then washed with PBS buffer for 3 times, and then incubated with horseradish peroxidase conjugated secondary antibody (Beijing Zhongsu Jin Qiao Biotechnology Co., Ltd.), and finally developed with diaminobenzidine (Beijing Zhongsu Jin Qiao Biotechnology Co., Ltd.).

[0066] (2) Survival analysis of patients according to immunohistochemical results: According to the total score of RBM4 staining, the patients were divided into two groups, RBM4 high expression group (174 cases) and low expression group (195 cases), and the survival curve was fitted according to the corresponding survival time (OS and DFS).

[0067] The survival analysis results are shown in Table 1: Figure 3 RBM4 high expression indicates better prognosis of renal cancer patients, which shows that the expression level of RBM4 has a significant positive correlation with the survival time of patients.

[0068] The above results indicate that RBM4 can be used as an effective biomarker for the diagnosis of renal cancer, and has application value for the clinical diagnosis of renal cancer.

[0069] Example 3 Prognostic value of TPM3P9 and RBM4 in renal cancer

[0070] 1. The same 369 clinical sample tissue chip of renal cancer patients (tissue chip from Sun Yat-Sen University Center for Cancer Prevention and Treatment) as in Example 2 was selected for TPM3P9 and RBM4 immunohistochemical detection, and the renal cancer patients were divided into four groups according to the TPM3P9 staining score: TPM3P9 high expression and RBM4 high expression, TPM3P9 low expression and RBM4 high expression, TPM3P9 high expression and RBM4 low expression, and TPM3P9 low expression and RBM4 low expression, and the survival curve (OS and DFS) was fitted according to the corresponding survival time of the patients for survival analysis. The specific steps are as follows:

[0071] (1) Preparation of anti-TPM3P9 polyclonal antibody

[0072] 1) The coding gene sequence of TPM3P9 protein (SEQ ID NO. 2) and pGEX-4T-1 plasmid (commonly available on the market) were digested with restriction endonuclease Xho1 and BamH1, and then ligated using T4 ligase to obtain recombinant plasmid pGEX-4T1-TPM3P9;

[0073] 2) The recombinant plasmid pGEX-4T1-TPM3P9 was transformed into E. coli BL21, which was cultured at 37°C for about 2 hours. When the OD 600=0.6, 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) was added to induce for 2-6 hours, and then the bacterial precipitate was collected by low-temperature centrifuge, resuspended and washed with PBS buffer for 3 times. The bacterial precipitate was repeatedly frozen and thawed for 3 times, and then ultrasonically broken, centrifuged at 12000 g for 30 minutes at 4°C, and the supernatant was reserved. 1 mL of GST-tag Purification Resin was added to the empty column of the affinity chromatography column, and then PBS buffer was passed through twice. The supernatant protein was added to the chromatography column loaded with GST-tag Purification Resin in multiple times. The bottom cover of the purification column was opened, and the effluent was collected. After the supernatant of the bacterial protein was filtered, PBS buffer was used to filter and wash away the impure proteins for 4 times. Eluent was prepared, and the final concentration was 10 mM GSH (glutathione) in 50 mM Tris-HCl solution, pH 8.0. Then the eluent was added, and the effluent was collected. The above liquid was concentrated by 10 kDa ultrafiltration tube, and finally washed with PBS buffer for 2 times. The purified fusion protein was cut by thrombin (4°C, 12 hours of enzyme cutting, 10 units of enzyme cutting 10 mg of fusion protein), and the cut protein solution was passed through GST-tag Purification Resin. The effluent was the protein encoded by the target gene, i.e., TPM3P9 protein.

[0074] 3) A Japanese white rabbit (female, 2.1 kg, from Wuhan Proteintech Group) was subcutaneously inoculated with 100 mg of TPM3P9 protein for immunization, and the rabbit produced anti-TPM3P9 antibody in vivo.

[0075] 4) The anti-TPM3P9 antibody was purified from the serum of the Japanese white rabbit (the purification was completed by Wuhan Proteintech Group).

[0076] (2) Immunohistochemical detection: the antibodies used were anti-TPM3P9 polyclonal antibody and anti-RBM4 polyclonal antibody, respectively, according to steps (1) and (2) of Example 2.

[0077] (3) Survival analysis of patients according to the immunohistochemical results: the renal cancer patients were divided into four groups according to the TPM3P9 staining score and RBM4 staining score: TPM3P9 high expression and RBM4 high expression, TPM3P9 low expression and RBM4 high expression, TPM3P9 high expression and RBM4 low expression, and TPM3P9 low expression and RBM4 low expression. The survival curves were fitted according to the corresponding survival time (OS and DFS).

[0078] The experimental results are shown in Table 1. Figure 4 Figure 4 ​A shows the immunohistochemical staining of TPM3P9 and RBM4 in 4 cases of renal cancer patients. Figure 4 B and Figure 4 C shows that the prognosis of patients in the four groups is different. Among them, the patients in the group with high expression of TPM3P9 and low expression of RBM4 have the worst prognosis, and the patients in the group with low expression of TPM3P9 and high expression of RBM4 have the best prognosis.

[0079] Example 4 Interaction of TPM3P9 and RBM4

[0080] Transfect TPM3P9-flag and RBM4-GFP plasmids in cells, lyse cells 48 hours after transfection and use cell lysate for immunoprecipitation experiment, and it is found that TPM3P9 and RBM4 have significant interaction. The specific steps are as follows:

[0081] (1) Construct pLVX-Puro-TPM3P9-flag on the skeleton of pLVX-Puro Vector (Addgene), and construct pEGFP-N1-RBM4 on the skeleton of pEGFP-N1 (Addgene).

[0082] 1) Synthesize the full sequence of RBM4 and TPM3P9-flag fusion sequence (commissioned to Aike Biological Company for synthesis), and the specific sequence is as follows:

[0083] RBM4-GFP sequence (SEQ ID NO. 3):

[0084]

[0085] TPM3P9-flag sequence (SEQ ID NO. 4):

[0086] GAATTCGCCACCGAACGAGCTGAGCTGGCAGAGTCCCGTTGCTGAGAGATGGATGAGCAGATCAGACTGATGGACCAGAACCTGAAGTGTCTGAGTGCAGCTGAAGAAAAGTACTCTCAAAAAGAAGACAAATGTGAGGAAGAGATGAAGATTCTTACTGATAATCTCAAGGAGGCAGAGACCCATGCTGAGTTGGCTGAGAGATCAGTAGCCAAGCTGGAAAAGACAATTGATGACTTGGAAGATAAACTGAAATGCACCAAAGAGGAACACCTCTGTACACAAAGGATGCTGGACCAGACTTTGCTTGACCTGAATGAGATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGTAGGGATCC.

[0087] 2) Then, the pLVX-Puro plasmid and the pEGPFP-N1 plasmid and the DNA of TPM3P9-flag and RBM4 were double digested with restriction enzymes EcoR I and BamH I at 37°C, and the enzyme-digested products were recovered using a DNA purification recovery kit (Tiangen Biotech).

[0088] 3) The enzyme-digested products were ligated using T4 DNA ligase at 16°C for 16 hours, and were transformed into DH5a competent cells, which were cultured at 37°C for 12 hours. Positive single clones were picked, amplified, and the plasmids were extracted to obtain the pLVX-Puro-TPM3P9-flag and pEGPFP-N1-RBM4 recombinant plasmids.

[0089] (2) The pLVX-Puro-TPM3P9-flag and pEGPFP-N1-RBM4 recombinant plasmids (1.5 μg each) were transfected into ACHN and 786-O cells (both purchased from ATCC) for 48 hours;

[0090] (3) Collect cells, wash with pre-cooled PBS three times, add appropriate amount of IP lysis buffer, lyse on ice for 30 minutes, then centrifuge at 4°C, 13200 rpm for 30 minutes, take 1 mg of protein in the supernatant, add 2.5 μg of IgG antibody, flag antibody and GFP antibody (Wuhan Proteintech Group Co., Ltd.) respectively, and incubate in a 4°C rotation mixer for 16 hours;

[0091] (4) Add 40 μL Protein A / G beads (Santa Cruz Biotechnology Co., Ltd.), and incubate in a 4°C rotation mixer for 4 hours;

[0092] (5) Centrifuge at 4°C, 2500 rpm for 5 minutes, discard the supernatant; resuspend the beads with 1 mL of IP lysis buffer, centrifuge at 4°C, 2500 rpm for 5 minutes, discard the supernatant, and repeat 3 times to remove impurities;

[0093] (6) Sample preparation: add 20 μL of 1x protein loading buffer to resuspend the beads, mix well, and boil in a boiling water bath for 10 minutes to denature the protein; the sample is used for Western blotting experiment.

[0094] The results of the Western blotting experiment are shown in Figure 5 , indicating that TPM3P9 protein has significant interaction with RBM4 protein, suggesting that TPM3P9 may regulate kidney cancer cell proliferation by interacting with RBM4.

[0095] Example 5 Influence of TPM3P9 and RBM4 on kidney cancer cell proliferation

[0096] First, respectively transfect pLVX-Puro Vector, pLVX-Puro-TPM3P9-flag, pLVX-Puro Vector and pLVX-Puro-RBM4 fragment, pLVX-Puro-TPM3P9-flag and pLVX-Puro-RBM4 in kidney cancer cells (ACHN and 786-O). In turn, obtain empty vector control cell strain (NC), TPM3P9 overexpressed cell strain (TPM3P9), overexpressed RBM4 empty vector control cell strain (NC+RBM4), and TPM3P9 overexpressed and RBM4 overexpressed cell strain (TPM3P9+RBM4), and then detect the proliferation ability of kidney cancer cells, and each experiment is set in triplicate, and the specific steps are as follows:

[0097] 1. Construction of recombinant plasmid and cell strain

[0098] 1) Construction of empty control cell strain (NC)

[0099] pLVX-Puro Vector (Addgene) and lentivirus packaging plasmids psPAX2, pMD2.G (Addgene) were transfected into 293T cells. After 24 and 48 hours, the cell supernatant was collected, filtered to remove cell debris, and the virus liquid was obtained. The virus liquid was then used to infect kidney cancer cells (ACHN and 786-O), and the cells were screened for resistance (2 μg / mL of puromycin) to obtain the empty control cell strain.

[0100] 2) Construction of TPM3P9 overexpression cell strain (TPM3P9)

[0101] The coding gene sequence of TPM3P9 protein (SEQ ID NO. 2) and pLVX-Puro Vector plasmid (Addgene) were digested with restriction enzymes EcoR I and BamH I, and then connected using T4 ligase to obtain the recombinant plasmid pLVX-Puro-TPM3P9. The recombinant plasmid pLVX-Puro-TPM3P9 was transfected into 293T cells together with lentivirus packaging plasmids psPAX2, pMD2.G (Addgene). After 24 and 48 hours, the cell supernatant was collected, filtered to remove cell debris, and the virus liquid was obtained. The virus liquid was then used to infect kidney cancer cells (ACHN and 786-O), and the cells were screened for resistance (2 μg / mL of puromycin) to obtain the TPM3P9 overexpression cell strain.

[0102] 3) Construction of empty vector control cell strain overexpressing RBM4 (NC+RBM4)

[0103] The coding gene sequence of RBM4 protein (Uniprot database accession number: Q9BWF3) and pLVX-Puro Vector plasmid (Addgene) were digested with restriction enzymes EcoR I and BamH I, and then connected using T4 ligase to obtain the recombinant plasmid pLVX-Puro-RBM4. The recombinant plasmid pLVX-Puro-RBM4 was transfected into 293T cells together with lentivirus packaging plasmids psPAX2, pMD2.G (Addgene). After 24 and 48 hours, the cell supernatant was collected, filtered to remove cell debris, and the virus liquid was obtained. The transfected cell supernatant was then removed, centrifuged, and filtered to remove cell debris. The resulting liquid was then used to infect kidney cancer cells (ACHN and 786-O) that stably expressed the empty control, and the cells were screened for resistance (2 μg / mL of puromycin) to obtain the RBM4 overexpression cell strain.

[0104] 4) Construction of TPM3P9 overexpression and RBM4 overexpression cell strain (TPM3P9+RBM4)

[0105] The coding gene sequence of RBM4 protein (uniprot database accession number: Q9BWF3) and pLVX-Puro Vector plasmid (Addgene) were digested by restriction enzymes EcoR I and BamH I, and then connected by T4 ligase to obtain recombinant plasmid pLVX-Puro-RBM4; the recombinant plasmid pLVX-Puro-RBM4 and lentivirus packaging plasmids (psPAX2, pMD2.G) were transfected into 293T cells, and after 24 hours and 48 hours, the cell supernatant was collected, filtered to remove cell debris, and virus liquid was obtained. Then the transfected cell supernatant was taken out, centrifuged and filtered to remove cell debris, and then it was used to infect the stable TPM3P9-overexpressing renal cancer cells (ACHN and 786-O) constructed in step 2) above, and the cells stably overexpressing TPM3P9 and RBM4 were obtained by resistance (2 μg / mL of puromycin) screening.

[0106] 2. CCK-8 experiment:

[0107] The empty vector control cell strain (NC), the TPM3P9-overexpressing renal cancer cell strain (TPM3P9), the empty vector control cell strain overexpressing RBM4 (NC+RBM4), and the cell strain overexpressing both TPM3P9 and RBM4 (TPM3P9+RBM4) constructed in this embodiment were digested to prepare a single cell suspension, and the cells were counted and inoculated into a 96-well plate at a density of 1000 cells per well and cultured at 37°C. At the end of each experiment, the original culture medium was discarded, and culture medium containing 10% CCK-8 reagent was added and incubated for 2 hours. Subsequently, the absorbance at 465 nm was detected, and the cell viability under different treatments was determined by comparing with the absorbance value of the untreated control. This experiment selected 0 hours, 24 hours, 48 hours, 72 hours and 96 hours for CCK-8 experiment.

[0108] 3. Mouse xenotransplantation experiment in vivo:

[0109] Twenty 6-week-old male NOD-SCID mice (purchased from Jiangsu Jizhu Pharmaceutical Company) were selected, and the empty vector control cell strain (NC), the TPM3P9-overexpressing renal cancer cell strain (TPM3P9), the empty vector control cell strain overexpressing RBM4 (NC+RBM4), and the cell strain overexpressing both TPM3P9 and RBM4 (TPM3P9+RBM4) constructed in this embodiment were used to construct a tumor xenotransplantation model:

[0110] (1) Each mouse was subcutaneously inoculated with 5×10 6A group of empty vector control ACHN cell lines (NC), TPM3P9 overexpressed renal cancer cell ACHN cell lines (TPM3P9), empty vector control ACHN cell lines overexpressing RBM4 (NC+RBM4), and ACHN cell lines overexpressing TPM3P9 and RBM4 (TPM3P9+RBM4), each group of 5 mice, to construct tumor xenograft models;

[0111] (2) Before the experiment, the NOD-SCID mice were anesthetized, and the degree of anesthesia was evaluated by painless and painful stimulation to determine that the mice were in an anesthetized state; the mice were subcutaneously injected with resuspended cells using a microsyringe with a 25G needle;

[0112] (3) Observe the growth of the mice, and remove the tumor at 30 days for subsequent study.

[0113] The results are shown in Figure 6 , as follows. Figure 6 The effects of TPM3P9 and RBM4 on the proliferation of ACHN and 786-O cells are shown, overexpression of TPM3P9 can promote the proliferation of renal cancer cells in vitro and in vivo, while overexpression of RBM4 can inhibit the proliferation of renal cancer cells, and overexpression of RBM4 in renal cancer cells overexpressing TPM3P9 can eliminate the promoting effect of TPM3P9 on the proliferation of renal cancer cells.

[0114] Example 6 Effect of Knocking Down TPM3P9 Protein on the Proliferation and Growth of Renal Cancer Cells

[0115] The results of the above Example 5 show that overexpression of RBM4 can inhibit the proliferation of renal cancer cells, and here the effect of knocking down TPM3P9 protein on the proliferation and growth of renal cancer cells is further studied.

[0116] 1. ACHN and 786-O cells with TPM3P9 knocked down by small interfering RNA, and verified by Western blotting, the specific steps are as follows:

[0117] (1) Commission Aikey Biological Company to synthesize TPM3P9 interfering RNA (siTPM3P9) and control interfering RNA (siNC), wherein the sequence of TPM3P9 interfering RNA is as follows: si-TPM3P9-sense: 5'-GGAAGAGAUGAAGAUUCUUTT-3'(SEQ ID NO. 5); si-TPM3P9-antisense: 5'-AAGAAUCUUCAUCUCUUCCTT-3'(SEQ ID NO. 6).

[0118] (2) Transient knockdown of TPM3P9: ACHN or 786-O cells were plated in 6-well plates, and when the cells reached a density of about 30-50%, 5 μL of control interference RNA (siNC) and TPM3P9 interference RNA (siTPM3P9) were taken, respectively, and were transiently transfected with Lipo3000 5 μL / tube; 48 hours after transfection, the cells were collected, and the knockdown effect was verified by Western blotting. The experiment was set up in triplicate.

[0119] Results are shown in Table 2: Figure 7 Results show that the TPM3P9 antibody prepared above can successfully detect the knockdown of TPM3P9 in renal cancer cell lines ACHN and 786-O.

[0120] 2. ACHN and 786-O cells stably knocked out of TPM3P9 were constructed by sgRNA targeting, and the knockdown effect was verified by Western blotting, with the specific steps as follows:

[0121] (1) Plasmid construction: The lentiCRISPR v2 (Addgene) plasmid was cut with restriction endonuclease Age I and EcoR I, and then the sgRNA#1 fragment (CACCGCATTCAGGTCAAGCAAAGTCGTTT) (SEQ ID NO. 7) and sgRNA#2 fragment (CACCGGCAGAGACCCATGCTGAGTGTTT) (SEQ ID NO. 8) (5'-3') (synthesized by Aigibi Biological Company) were connected to the cut lentiCRISPR v2, respectively, to construct the recombinant plasmid (sg#1, sg#2) for knocking down TPM3P9.

[0122] (2) Stable cell line construction: 293T cells were plated in a six-well plate, and when the cell density reached about 80%, 1 μg of control sg NC (Addgene) and recombinant plasmid (sg#1, sg#2) and lentivirus packaging plasmid (psPAX2, pMD2.G) were used to transfect the cells in the cell dish with Lipo3000 4 μL / tube; 48 hours after transfection, the virus supernatant was collected and filtered with a 0.45 μM filter to remove cell debris, etc., and then the virus was added to the target ACHN and 786-O cells. After 48 hours of virus infection, the liquid was changed, and then 2 μg / mL of puromycin was added for screening of ACHN and 786-O overexpression positive cells, and the puromycin was removed after the cell number did not change significantly.

[0123] (3) Western blotting experiment to detect the knockdown effect: The cells were collected, and the knockdown effect was verified by Western blotting. The successfully constructed cell strains were frozen and stored in a -80°C liquid nitrogen tank.

[0124] (4) Observe the effect of stable knockdown of TPM3P9 protein on the proliferation of renal cancer cells in vitro by CCK-8 experiment, colony formation experiment and EdU experiment.

[0125] The results are shown in Figure 8 : The results show that TPM3P9 protein in ACHN and 786-O cells is successfully knocked down Figure 8 (A); CCK-8 Figure 8 (B), colony formation Figure 8 (C) and EdU experiment Figure 8 (D) all show that ACHN and 786-O cells stably knocked down TPM3P9 protein can significantly inhibit the proliferation and colony formation ability of renal cancer cells.

[0126] 3. Stable knockdown of TPM3P9 protein inhibits tumor proliferation of renal cancer cells in mice. The specific experimental method is as follows:

[0127] Select 15 6-week-old male NOD-SCID mice (purchased from Jiangsu Jicai Yekang Company) and use the above constructed knockdown control ACHN cell line (control group, sg NC) and TPM3P9 knockdown ACHN cell line (experimental group, sg#1 and sg#2) to construct tumor xenograft model:

[0128] (1) Each mouse was subcutaneously inoculated with 5x10 6 knockdown control ACHN cells (control group, sg NC) and its corresponding TPM3P9 knockdown ACHN cells (experimental group, sg#1 and sg#2), 5 mice in each group, to construct tumor xenograft model;

[0129] (2) Before the experiment, the NOD-SCID mice were anesthetized, and the degree of anesthesia was evaluated by painless and painful stimulation to determine that the mice were in an anesthetized state; the mice were subcutaneously injected with resuspended cells using a 25G needle microsyringe;

[0130] (3) Observe the growth of the mice, and remove the tumor at 30 days for further study.

[0131] The results are shown in Figure 9 : The subcutaneous tumor experiment in mice showed that knockdown of TPM3P9 could inhibit the subcutaneous growth of renal cancer cells in mice Figure 9 (A and Figure 9 B), knockdown of TPM3P9 significantly reduced the size and weight of subcutaneous tumors in mice Figure 9 (C), while the body weight of mice in each treatment group did not show significant difference Figure 9D) In vivo experiments show that knocking down TPM3P9 can inhibit the proliferation and growth ability of renal cancer cells.

[0132] The above embodiments are preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.

Claims

1. The use of an immunohistochemical reagent for detecting the expression levels of TPM3P9 protein and RBM4 protein in the preparation of a product for predicting the prognosis risk of kidney cancer, characterized in that: The amino acid sequence of the TPM3P9 protein is shown as SEQ ID NO.

1.

2. The use according to claim 1, wherein: The coding gene sequence of the TPM3P9 protein is any one of the following sequences: (a) the accession number of NCBI is NR_003148.3; (b) shown as SEQ ID NO.

2.

3. Use according to any one of claims 1 to 2, characterized in that: The product includes a kit or a chip.

4. Application of the TPM3P9 protein and the RBM4 protein as a drug target in the preparation of a drug for treating kidney cancer and / or resisting the proliferative growth of kidney cancer cells, characterized in that: The treatment of kidney cancer or the anti-proliferation and growth of kidney cancer cells is achieved by knocking down the TPM3P9 protein and overexpressing the RBM4 protein.

5. Use according to claim 4, characterized in that: The knocking down of the TPM3P9 protein is achieved by silencing or knocking out the TPM3P9 gene, wherein, The siRNA sequence for silencing the TPM3P9 gene is: si-TPM3P9-sense: 5'-GGAAGAGAUGAAGAUUCUUTT-3'; si-TPM3P9-antisense: 5'-AAGAAUCUUCAUCUCUUCCTT-3'; The sgRNA sequence for targeted knockout of the TPM3P9 gene is any one of the following sequences: sgRNA #1: 5'-CACCGCATTCAGGTCAAGCAAAGTCGTTT-3'; sgRNA #2: 5'-CACCGGCAGAGACCCATGCTGAGTGTTT-3'.

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