Application of TPM3P9 protein and RBM4 protein in kidney cancer

Abstract

The invention discloses an application of a TPM3P9 protein and an RBM4 protein in kidney cancer. According to the application disclosed by the invention, high expression of the RBM4 indicates better prognosis of a renal cancer patient, and the prognosis of the patient with low expression of the TPM3P9 and high expression of the RBM4 is the best, so that the RBM4 protein or the RBM4 protein and the TPM3P9 protein can be used as a clinical diagnosis or prognosis biomarker of the renal cancer. It is also found through experiments that proliferation and growth of kidney cancer cells can be regulated and controlled by regulating and controlling the expression quantity of the TPM3P9 protein, and therefore the RBM4 protein and the TPM3P9 protein can be combined to be used for developing drugs for treating kidney cancer or proliferation and growth of kidney cancer cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the application of TPM3P9 protein and RBM4 protein in renal cancer. Background Art

[0002] Renal cancer is a common malignant tumor of the urinary system. Its morbidity and mortality have shown an increasing trend in recent years, seriously endangering human life safety. Since the early symptoms of renal cancer are not obvious, most renal cancer patients have developed to the middle and late stages when they are clinically diagnosed, missing the best time for treatment. Local renal cancer can be treated by radical nephrectomy and cryoablation. Although nephrectomy has a significant effect on the treatment of local renal cancer, more than 15% of renal cancer patients will have distant metastasis, which requires systemic treatment and is accompanied by a high mortality rate. The 5-year survival rate of patients with metastatic renal cancer is less than 10%. Renal cancer is insensitive to traditional chemotherapy and radiotherapy. The current drug treatment for renal cancer is mainly based on targeted therapy of vascular endothelial growth factor (VEGF) and mTOR pathways, but the treatment effect is still not very ideal. Therefore, it is imperative to further explore renal cancer biomarkers and therapeutic targets 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 some of the non-coding genes that account for about 98% of the human genome can encode new proteins. These new proteins are usually translated from small open reading frames (sORFs) on lncRNA and play an important regulatory role in tumor biology. Mass spectrometry technology is considered to be the most direct tool for the identification of new proteins, but mass spectrometry technology relies on a reference database established by protein sequences. Translationomics (RNC-seq and Ribo-seq) can discover RNA with translation function in cells on a large scale, making up for the lack of new protein databases in mass spectrometry technology. Therefore, the combined use of proteomics and translationomics technologies will help the discovery and identification of new proteins.

[0004] RBM4 protein is a tumor suppressor protein that can inhibit the target gene by regulating the alternative splicing of pre-mRNA. TPM3P9 (Homo sapiens tropomyosin 3pseudogene 9) is a long non-coding RNA. There are still no relevant reports on the functions of TPM3P9 and RBM4 proteins in the human body and their relationship with renal cancer. Summary of the invention

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

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

[0007] Another object of the present invention is to provide a use of TPM3P9 protein and RBM4 protein in combination in the preparation of a drug for treating renal cancer and / or inhibiting the proliferation and growth of renal cancer cells.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] 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 renal cancer, and / or a product for predicting the prognosis risk of renal 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] Use of reagents for detecting the expression levels of TPM3P9 protein and RBM4 protein in the preparation of products for diagnosing or assisting in the diagnosis of renal cancer, and / or products for predicting the prognosis risk of renal cancer.

[0012] The amino acid sequence of the TPM3P9 protein is shown in 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 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 levels of TPM3P9 protein and RBM4 protein may be a reagent for detecting TPM3P9 protein and RBM4 protein respectively, or a reagent for detecting TPM3P9 protein and RBM4 protein simultaneously.

[0018] The products include test kits, test strips or chips.

[0019] The invention relates to the use of TPM3P9 protein (as a target) and RBM4 protein in combination in the preparation of a drug for treating renal cancer and / or inhibiting the proliferation and growth of renal cancer cells.

[0020] The treatment of renal cancer or the prevention of renal cancer cell proliferation is achieved by knocking down the TPM3P9 protein and overexpressing the RBM4 protein; knocking down the TPM3P9 protein can inhibit the proliferation and growth ability of renal cancer cells (inhibiting the cloning of renal cancer cells), while overexpressing the RBM4 protein can also inhibit the proliferation and growth ability of renal cancer cells.

[0021] The drug is preferably administered by subcutaneous injection or intravenous injection.

[0022] The renal cancer includes renal clear cell carcinoma and the like.

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

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

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

[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 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 RBM4 protein is preferably achieved by the following method:

[0032] (1) The coding gene sequence of RBM4 protein and the pLVX-Puro Vector plasmid were digested with restriction endonucleases EcoR I and BamH I, and then ligated with T4 ligase to obtain the recombinant plasmid pLVX-Puro-RBM4;

[0033] (2) The recombinant plasmid pLVX-Puro-RBM4 and the packaging plasmid were transfected into 293T cells, and the supernatant of the transfected cells was removed, centrifuged, and filtered to remove cell debris. The supernatant was then used to infect renal cancer cells, and a cell line overexpressing the RBM4 protein was obtained after resistance screening.

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

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

[0036] The present invention combines cell proliferation phenotype and mouse in vitro xenograft experiments to find that high expression of the new protein TPM3P9 promotes the proliferation of renal cancer cells, while high expression of the RBM4 protein inhibits the proliferation of renal cancer cells. Mechanistically, the cancer-promoting protein TPM3P9 interacts with the splicing factor RBM4 and regulates the growth and proliferation of renal cancer cells by regulating alternative splicing. Therefore, clinically targeting TPM3P9 and RBM4 proteins can inhibit the proliferation of renal cancer cells.

[0037] Compared with the prior art, the present invention has the following advantages and effects:

[0038] 1. The present invention discovered for the first time that the TPM3P9 protein encoded by lncRNA is related to the proliferation of renal cancer. As a cancer-promoting protein, the TPM3P9 protein can effectively promote the growth and proliferation of renal 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 renal cancer. Therefore, TPM3P9 and RBM4 can be used as regulatory targets for cancer cell proliferation.

[0039] 2. The present invention found that the proliferation and growth of tumors can be regulated by regulating the expression of TPM3P9 protein. In renal cancer, the tumor-promoting protein TPM3P9 interacts with the splicing factor RBM4, regulates alternative splicing and then regulates the growth and proliferation of renal cancer cells. Therefore, 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 the clinical diagnosis of renal cancer, and for the development of drugs for the treatment of renal cancer or the proliferation and growth of renal cancer cells.

[0040] 3. The present invention found that high expression of RBM4 indicates a better prognosis for patients with renal cancer. Therefore, RBM4 protein can be used as an effective biomarker for clinical diagnosis of renal cancer. At the same time, the present invention also found that the patients with high expression of TPM3P9 and low expression of RBM4 had the worst prognosis, while the patients with low expression of TPM3P9 and high expression of RBM4 had the best prognosis. Therefore, TPM3P9 and RBM4 can be used together as biomarkers for clinical diagnosis or prognosis of renal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0042] Figure 2 These are experimental results of the role of RBM4 in the prognosis of renal cancer; A shows that the expression of RBM4 in renal cancer is lower than that in adjacent tissues; B shows that the expression of RBM4 is reduced in high-stage renal cancer samples; C shows that the expression of RBM4 in metastatic renal cancer is lower than that in non-metastatic renal cancer; and D shows that high expression of RBM4 is associated with a good prognosis of renal cancer.

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

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

[0045] Figure 5These are the results of a protein immunoblotting experiment on the interaction between TPM3P9 and RBM4; A is TPM3P9 immunoprecipitated with RBM4; B is RBM4 immunoprecipitated with TPM3P9; C shows that overexpression of TPM3P9 protein in ACHN and 786-O cells does not affect the protein expression of RBM4.

[0046] Figure 6 The figure is the effect of TPM3P9 and RBM4 on the proliferation of renal cancer cells; 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 effects of TPM3P9 and RBM4 on the growth of renal cancer cells in mice; E is the weight change of mice during the in vivo experiment.

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

[0048] Figure 8 The figure shows the effect of TPM3P9 protein on the proliferation and growth of renal cancer cells in vitro; A shows the successful knockdown of TPM3P9 protein in renal cancer cells; B shows the ability of silencing TPM3P9 protein to inhibit the growth of renal cancer cells (CCK-8 experiment); C shows the ability of knocking down TPM3P9 protein to inhibit the cloning ability of renal cancer cells; D shows the ability of knocking down TPM3P9 protein to inhibit the proliferation of renal cancer cells (EdU experiment).

[0049] Fig. 9 The figure shows the effect of TPM3P9 protein on the proliferation and growth of renal cancer cells in mice; A is the growth curve of renal cancer cells in mice inhibited by knocking down TPM3P9 protein; B is the growth curve of renal cancer cells in mice inhibited by knocking down TPM3P9 protein; C is the weight of subcutaneous tumors in mice inhibited by knocking down TPM3P9 protein; D is the weight change of mice during the in vivo experiment. DETAILED DESCRIPTION

[0050] The present invention will be described in further detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples 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 present invention can be obtained commercially.

[0051] Example 1 Identification of the new protein TPM3P9

[0052] 1. The present invention discovered that TPM3P9 (Homo sapiens tropomyosin 3pseudogene 9, long non-coding RNA) (NCBI accession number: NR_003148.3, 2932bp) can encode a new protein with a coding length of 92 amino acids, which is named TPM3P9 protein; wherein,

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

[0054] MDEQIRLMDQNLKCLSAAEEKYSQKEDKCEEEMKILTDNLKEAETHAELAERSVAKL EKTIDDLEDKLKCTKEEHLCTQRMLDQTLLDLNEM;

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

[0056] ATGGATGAGCAGATCAGACTGATGGACCAGAACCTGAAGTGTCTGAGTGCAGCTGAAGAAAAGTACTCTCAAAAAGAAGACAAATGTGAGGAAGAGATGAAGATTCTTACTGATAATCTCAAGGAGGCAGAGACCCATGC TGAGTTGGCTGAGAGATCAGTAGCCAAGCTGGAAAAGACAATTGATGACTTGGAAGATAAACTGAAATGCACCAAAGAGGAACACCTCTGTACACAAAGGATGCTGGACCAGACTTTGCTTGACCTGAATGAGATGTAG.

[0057] The coding sequence of TPM3P9 is as Figure 1 As shown in A, a 10 kDa protein is encoded by the non-coding RNA-TPM3P9.

[0058] 2. This experiment further provides protein evidence for TPM3P9 identification by mass spectrometry. The specific experimental steps are as follows:

[0059] (1) Protein enzymatic hydrolysis: Renal cancer cell line ACHN and 786-O cell samples (purchased from ATCC) were lysed to obtain protein solution, and then 200 μg of protein was taken for enzymatic hydrolysis. After the protein was reduced and alkylated, mass spectrometry grade trypsin (Beijing Shengxia Protein) was added at a ratio of 1:30 to 40 for protein, and the solution was hydrolyzed at 37°C for 16 hours. Finally, the peptides obtained by enzymatic hydrolysis were freeze-dried in a vacuum dryer.

[0060] (2) Desalting: The lyophilized enzymatic peptides were re-dissolved in mass spectrometry grade water (ThermoFisher Scientific) containing 0.5% (v / v) TFA (trifluoroacetic acid). After the concentration was detected, 15 μg of the enzymatic peptides were desalted by passing through a C18 column (Thermo Fisher Scientific). Finally, the desalted peptides were lyophilized by passing through a vacuum dryer.

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

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

[0063] 1. Analyze the renal cancer and adjacent cancer data in the TCGA (The Cancer Genome Atlas, https: / / portal.gdc.cancer.gov / ) dataset. Figure 2 As shown: It was found that the expression of RBM4 in renal cancer tissues was significantly lower than that in adjacent tissues ( Figure 2 A), and RBM4 expression was lower in patients with higher T stages of renal cancer (divided into T1 (I), T2 (II), T3 (III), and T4 (IV)) ( Figure 2 B), RBM4 expression is lower in patients with metastatic renal cell carcinoma (metastatic patients (N1), non-metastatic patients (N0)) Figure 2 C), high expression of RBM4 contributes to a good prognosis in renal cancer ( Figure 2 D), which indicates that RBM4 plays a tumor suppressor function in renal cancer.

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

[0065] (1) Immunohistochemical detection: The tissue sample chips and tissue samples were incubated with anti-RBM4 polyclonal antibody (Wuhan Proteintech Group) at a dilution of 1:500 (volume ratio) at 4°C overnight, then washed three times with PBS buffer, incubated with horseradish peroxidase-conjugated secondary antibody (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.), and finally developed with diaminobenzidine (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.).

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

[0067] The survival analysis results are as follows Figure 3 As shown: High expression of RBM4 indicates a better prognosis for patients with renal cancer, indicating that there is a significant positive correlation between the expression level of RBM4 and 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 in the clinical diagnosis of renal cancer.

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

[0070] 1. Tissue chips of 369 clinical samples of renal cancer patients were selected as in Example 2 (the tissue chips were from the Cancer Prevention and Treatment Center of Sun Yat-sen University) for TPM3P9 and RBM4 immunohistochemistry detection, and the renal cancer patients were divided into four groups according to the TPM3P9 staining score: high expression of TPM3P9 and high expression of RBM4, low expression of TPM3P9 and high expression of RBM4, high expression of TPM3P9 and low expression of RBM4, and low expression of TPM3P9 and low expression of RBM4. The survival analysis was performed by fitting the survival curve (OS and DFS) according to the corresponding survival time of the patients. 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 in the market) were digested with restriction endonucleases Xho1 and BamH1, and then ligated with T4 ligase to obtain the recombinant plasmid pGEX-4T1-TPM3P9;

[0073] 2) Transform the recombinant plasmid pGEX-4T1-TPM3P9 into E. coli BL21 and culture in a 37°C constant temperature shaker for about 2 hours. 600= 0.6, add 0.5mM isopropyl-β-D-thiogalactoside IPTG to induce for 2 to 6 hours. After the induction, collect the precipitate of the bacteria by centrifugation in a low-temperature centrifuge, and resuspend and wash 3 times with PBS buffer. Repeat freezing and thawing 3 times, then ultrasonically disrupt the bacteria, centrifuge at 4°C, 12000g for 30 minutes, and retain the supernatant. Add 1mL of GST-tag Purification Resin to the empty column tube of the affinity chromatography column and pass it twice with PBS buffer. Add the supernatant protein to the chromatography column loaded with GST-tag Purification Resin in multiple times. Open the bottom cover of the purification column and let the liquid flow out. After the bacterial protein supernatant is completely filtered, add PBS buffer and filter 4 times to wash away the impurities. Prepare the eluent, a 50mM Tris-HCl solution with a final concentration of 10mMGSH (glutathione), pH 8.0. Then add the eluent and collect the liquid that flows out. The above liquid was concentrated by a 10kDa ultrafiltration tube, and finally washed twice with PBS buffer. The purified fusion protein was digested with thrombin (4°C, digestion for 12 hours, 10 units of enzyme digestion for 10 mg of fusion protein), and the protein solution after digestion was passed through GST-tag Purification Resin. The liquid flowing out was the protein encoded by the target gene, namely TPM3P9 protein.

[0074] 3) Japanese white rabbits (Wuhan Proteintech Group, female, 2.1 kg) were subcutaneously inoculated with 100 mg of TPM3P9 protein for immunization, and anti-TPM3P9 antibodies were produced in the rabbits;

[0075] 4) Anti-TPM3P9 antibody was purified from Japanese white rabbit serum (commissioned by Wuhan Proteintech Group).

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

[0077] (3) Survival analysis of patients was performed based on the results of immunohistochemistry: Combined with the TPM3P9 staining score, renal cancer patients were divided into four groups: high expression of TPM3P9 and high expression of RBM4, low expression of TPM3P9 and high expression of RBM4, high expression of TPM3P9 and low expression of RBM4, and low expression of TPM3P9 and low expression of RBM4. The survival curve was fitted based on the corresponding survival time (OS and DFS).

[0078] The experimental results are as follows Figure 4 As shown: Figure 4A shows the immunohistochemical staining of TPM3P9 and RBM4 in 4 patients with renal cancer. 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, while the patients in the group with low expression of TPM3P9 and high expression of RBM4 have the best prognosis.

[0079] Example 4 Interaction between TPM3P9 and RBM4

[0080] TPM3P9-flag and RBM4-GFP plasmids were transfected into cells, cells were lysed 48 hours after transfection and immunoprecipitation experiments were performed using cell lysate. The results showed that TPM3P9 and RBM4 had significant interactions. The specific steps are as follows:

[0081] (1) The recombinant plasmids pLVX-Puro-TPM3P9-flag were constructed on the backbone of pLVX-Puro Vector (Addgene), and the recombinant plasmids pEGPFP-N1-RBM4 were constructed on the backbone of pEGPFP-N1 (Addgene).

[0082] 1) The fusion sequence of RBM4 and TPM3P9-flag was synthesized in full sequence (commissioned by Aiki Biotech), the specific sequence is as follows:

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

[0084]

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

[0086] GAATTCGCCACCGAACGAGCTGAGCTGGCAGAGTCCCGTTGCTGAGAGATGGATGAGCAGATCAGACTGATGGACCAGAACCTGAAGTGTCTGAGTGCAGCTGAAGAAAAGTACTCTCAAAAAGAAGACAAATGTGAGGAAGAGATGAAGATTCTTACTGATAATCTCAAGGAGGCAGAGACCCATGCTGAGTTGGCTGA GAGATCAGTAGCCAAGCTGGAAAAGACAATTGATGACTTGGAAGATAAACTGAAATGCACCAAAGAGGAACACCTCTGTACACAAAGGATGCTGGACCAGACTTTGCTTGACCTGAATGAGATGGACTACAAAGACCATGACGGTGATTATAAAAGATCATGACATCGATTACAAGGATGACGATGACAAGTAGGGATCC.

[0087] 2) Then, the pLVX-Puro plasmid and pEGPFP-N1 plasmid as well as the DNA of TPM3P9-flag and RBM4 were double-digested at 37°C using restriction endonucleases EcoR I and BamH I, and the DNA was recovered by enzyme digestion using a DNA purification and recovery kit (Tiangen Biotechnology).

[0088] 3) Use T4 DNA ligase to connect the digested products at 16°C for 16 hours. Transform into DH5α competent cells and culture at 37°C for 12 hours. Pick the positive single clones, amplify them, and extract the plasmids to obtain the pLVX-Puro-TPM3P9-flag and pEGPFP-N1-RBM4 recombinant plasmids;

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

[0090] (3) The cells were collected and washed three times with pre-cooled PBS, and then lysed on ice for 30 min with an appropriate amount of IP lysis buffer. The cells were then centrifuged at 4°C and 13200 rpm for 30 min. 1 mg of protein in the supernatant was added with 2.5 μg of IgG antibody, flag antibody, and GFP antibody (Wuhan Proteintech Group), respectively, and incubated in a rotating mixer at 4°C for 16 h.

[0091] (4) Add 40 μL of Protein A / G beads (Santa Cruz Biotechnology) and incubate at 4°C in a rotating mixer for 4 h;

[0092] (5) Centrifuge at 2500 rpm for 5 min at 4°C and discard the supernatant; gently resuspend the beads in 1 mL of IP lysis buffer and centrifuge at 2500 rpm for 5 min at 4°C and discard the supernatant. Repeat three times to remove impurities;

[0093] (6) Sample preparation: Add 20 μL of 1× 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 protein immunoblotting experiments.

[0094] The results of immunoblotting experiments were Figure 5 As shown, there is a significant interaction between TPM3P9 protein and RBM4 protein, suggesting that TPM3P9 may regulate the proliferation of renal cancer cells by interacting with RBM4.

[0095] Example 5 Effects of TPM3P9 and RBM4 on the proliferation of renal cancer cells

[0096] First, pLVX-Puro Vector, pLVX-Puro-TPM3P9-flag, pLVX-Puro Vector and pLVX-Puro-RBM4 fragment, pLVX-Puro-TPM3P9-flag and pLVX-Puro-RBM4 were transfected into renal cancer cells (ACHN and 786-O) respectively. The empty vector control cell line (NC), TPM3P9 overexpression cell line (TPM3P9), RBM4 overexpression empty vector control cell line (NC+RBM4), and TPM3P9 overexpression and RBM4 overexpression cell line (TPM3P9+RBM4) were obtained in turn, and then the proliferation ability of renal cancer cells was detected. The experiments were repeated three times. The specific steps are as follows:

[0097] 1. Construction of recombinant plasmids and cell lines

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

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

[0100] 2) Construction of TPM3P9 overexpressing cell line (TPM3P9)

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

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

[0103] The coding gene sequence of RBM4 protein (accession number of Uniprot database: Q9BWF3) and pLVX-PuroVector plasmid (Addgene) were digested with restriction enzymes EcoR I and BamH I, and then ligated with T4 ligase to obtain recombinant plasmid pLVX-Puro-RBM4; the recombinant plasmid pLVX-Puro-RBM4 and lentiviral packaging plasmids psPAX2 and pMD2.G (Addgene) were transfected into 293T cells, and after 24 hours and 48 hours, the cell supernatant was collected and filtered to remove cell debris to obtain virus liquid. Then the supernatant of the transfected cells was taken out, centrifuged, filtered to remove cell debris, and then infected with renal cancer cells (ACHN and 786-O) stably expressing empty control, and RBM4 overexpression cell lines were obtained by resistance (2μg / mL puromycin) screening.

[0104] 4) Construction of TPM3P9-overexpressing and RBM4-overexpressing cell lines (TPM3P9+RBM4)

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

[0106] 2. CCK-8 experiment:

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

[0108] 3. Xenograft experiment in mice:

[0109] Twenty 6-week-old male NOD-SCID mice (purchased from Jiangsu Jicui Pharmaceutical Co., Ltd.) were selected, and the empty vector control cell line (NC) constructed in this example, the renal cancer cell line overexpressing TPM3P9 (TPM3P9), the empty vector control cell line overexpressing RBM4 (NC+RBM4), and the cell line overexpressing TPM3P9 and RBM4 (TPM3P9+RBM4) were used to construct a tumor xenograft model:

[0110] (1) Each mouse was subcutaneously inoculated with 5×10 6The tumor xenograft model was established by using 5 mice in each group: empty vector control ACHN cell line (NC), TPM3P9 overexpressing renal cancer cell line ACHN cell line (TPM3P9), empty vector control ACHN cell line overexpressing RBM4 (NC+RBM4), and TPM3P9 overexpressing and RBM4 overexpressing ACHN cell line (TPM3P9+RBM4);

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

[0112] (3) Observe the growth status of mice and remove the tumors at 30 days for follow-up studies.

[0113] The results are as follows Figure 6 As shown, Figure 6 The effects of TPM3P9 and RBM4 on the proliferation of ACHN and 786-O cells were demonstrated. Overexpression of TPM3P9 can promote the proliferation of renal cancer cells in vivo and in vitro, while overexpression of RBM4 can inhibit the proliferation of renal cancer cells. Overexpression of RBM4 in renal cancer cells overexpressing TPM3P9 can eliminate the promoting effect of TPM3P9 on the proliferation ability 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 Example 5 above indicate that overexpression of RBM4 will inhibit the proliferation of renal cancer cells. Here, the effect of knocking down TPM3P9 protein on the proliferation and growth of renal cancer cells is further studied.

[0116] 1. TPM3P9 was knocked down in ACHN and 786-O cells by small interfering RNA and verified by western blot. The specific steps are as follows:

[0117] (1) Entrust Aiki Biotechnology Co., Ltd. to synthesize TPM3P9 interfering RNA (siTPM3P9) and control interfering RNA (si NC), wherein the sequence of TPM3P9 interfering RNA is as follows: si-TPM3P9-sense: 5′-GGAAGAGAUGAAGA UUCUUTT-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. When the cells grew to a density of about 30-50%, 5 μL of control interfering RNA (siNC) and TPM3P9 interfering RNA (siTPM3P9) were taken respectively and transiently transfected with Lipo3000 5 μL / tube; 48 hours after transfection, cells were collected and the knockdown effect was verified by immunoblotting. The experiment was repeated three times.

[0119] The results are as follows Figure 7 As shown: The results showed that the TPM3P9 antibody prepared as described above could successfully detect the knockdown of TPM3P9 in renal cancer cell lines ACHN and 786-O.

[0120] 2. Stable knockout of TPM3P9 was constructed by targeting knockout of TPM3P9 by sgRNA and ACHN and 786-O cells, and verified by western blotting. The specific steps are as follows:

[0121] (1) Plasmid construction: The lentiCRISPR v2 (addgene) plasmid was cut with restriction endonucleases AgeI and EcoR I, and then the sgRNA#1 fragment (CACCGCATTCAGGTCAAGCAAAGTCGTTT) (SEQ ID NO.7) and the sgRNA#2 fragment (CACCGGCAGAGACCCATGCTGAGTGTTT) (SEQ ID NO.8) (5′-3′) (commissioned to Addgene for synthesis) were ligated to the cut lentiCRISPR v2 using T4 DNA ligase to construct recombinant plasmids (sg#1, sg#2) for knocking down TPM3P9.

[0122] (2) Construction of stable cell lines: 293T cells were plated in six-well plates. When the cell density reached about 80%, 1 μg of each of the control group sg NC (Addgene), recombinant plasmids (sg#1, sg#2) and lentiviral packaging plasmids (psPAX2, pMD2.G) were transfected into the cell dishes using Lipo3000 4 μL / tube. 48 hours after transfection, the viral 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 viral infection, the medium was changed, and then 2 μg / mL of puromycin was added for the screening of ACHN and 786-O overexpression positive cells, and puromycin was withdrawn after there was no significant change in the cell number.

[0123] (3) Western blotting to detect knockdown effect: Collect cells and verify the knockdown effect by Western blotting. The successfully constructed cell lines are frozen and stored in a -80°C liquid nitrogen tank.

[0124] (4) The effect of stable knockdown of TPM3P9 protein on the proliferation and growth of renal cancer cells in vitro was observed through CCK-8 experiment, clone formation experiment and EdU experiment.

[0125] The results are as follows Figure 8 As shown: The results showed that TPM3P9 protein was successfully knocked down in ACHN and 786-O cells ( Figure 8 A); CCK-8( Figure 8 B) Clone formation ( Figure 8 C) and EdU experiments ( Figure 8 D) Both showed that ACHN and 786-O cells with stable knockdown of TPM3P9 protein could significantly inhibit the proliferation and clone formation ability of renal cancer cells.

[0126] 3. Experiment on in vivo tumor proliferation inhibition of renal cancer cells in mice with stable knockout of TPM3P9 protein. The specific experimental methods are as follows:

[0127] Fifteen 6-week-old male NOD-SCID mice (purchased from Jiangsu Jicui Pharmaceutical Co., Ltd.) were selected and the tumor xenograft model was constructed using the above-constructed knockout control ACHN cell line (control group, sg NC) and TPM3P9 knockout ACHN cell line (experimental groups, sg#1 and sg#2):

[0128] (1) Each mouse was subcutaneously inoculated with 5×10 6 ACHN cells with knockout control (control group, sg NC) and their corresponding ACHN cells with TPM3P9 knockout (experimental groups, sg#1 and sg#2), 5 mice in each group, were used to construct tumor xenograft models;

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

[0130] (3) Observe the growth status of mice and remove the tumors at 30 days for follow-up studies.

[0131] The results are as follows Fig. 9 As shown: Mouse subcutaneous tumor formation experiments showed that knocking down TPM3P9 can inhibit the subcutaneous growth of renal cancer cells in mice ( Fig. 9 A and Fig. 9 B), knockdown of TPM3P9 significantly reduced the size and weight of subcutaneous tumors in mice ( Fig. 9 C), while there was no significant difference in the body weight of mice in each treatment group during the experiment ( Fig. 9D). In vivo experiments showed that knocking down TPM3P9 could inhibit the proliferation and growth of renal cancer cells.

[0132] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. 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 renal cancer, and / or a product for predicting the prognosis risk of renal cancer.

2. Use of a reagent for detecting the expression levels of TPM3P9 protein and RBM4 protein in the preparation of a product for diagnosing or assisting in the diagnosis of renal cancer, and / or a product for predicting the prognosis risk of renal cancer, characterized in that: The amino acid sequence of the TPM3P9 protein is shown in SEQ ID NO.

1.

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

2.

4. The use according to any one of claims 1 to 3, characterized in that: The products include test kits, test strips or chips.

5. Use of TPM3P9 protein and RBM4 protein in combination in the preparation of drugs for treating renal cancer and / or inhibiting the proliferation and growth of renal cancer cells.

6. The use according to claim 5, characterized in that: The treatment of renal cancer or the inhibition of renal cancer cell proliferation and growth is achieved by knocking down TPM3P9 protein and overexpressing RBM4 protein.

7. The use according to claim 6, characterized in that: The knockdown of TPM3P9 protein is achieved by silencing or knocking out the TPM3P9 gene, wherein: The siRNA sequence used to silence the TPM3P9 gene is: si-TPM3P9-sense:5′-GGAAGAGAUGAAGAUUCUUTT-3′; si-TPM3P9-antisense:5′-AAGAAUCUUCAUCUCUUCCTT-3′; The sgRNA sequence used for targeted knockout of the TPM3P9 gene is any of the following sequences: sgRNA#1: 5′-CACCGCATTCAGGTCAAGCAAAGTCGTTT-3′; sgRNA#2: 5′-CACCGGCAGAGACCCATGCTGAGTGTTT-3′.

8. The use according to claim 6, characterized in that: The drug for resisting the proliferation and growth of renal cancer cells includes a drug for regulating the alternative splicing function of renal cancer cells.

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