TPM3P9 protein coded by lncRNA and application of TPM3P9 protein in tumor diagnosis and treatment

Abstract

The invention discloses a TPM3P9 protein coded by lncRNA (long non-coding ribonucleic acid) and application of the TPM3P9 protein in tumor diagnosis and treatment. The invention finds that long-chain non-coding RNA-TPM3P9 (NCBI accession number: NR003148.3) can code a new protein, the coding length is 92 amino acids (SEQ ID NO.1), the long-chain non-coding RNA-TPM3P9 is named as TPM3P9 protein, and the coding gene sequence of the long-chain non-coding RNA-TPM3P9 is as shown in SEQ ID NO.2. The invention also finds that the TPM3P9 protein is obviously differentially expressed in kidney cancer tissues and para-carcinoma tissues, so that the TPM3P9 protein can be used as a biomarker for clinical diagnosis of kidney cancer; meanwhile, the invention also finds that the multiplication capacity and the clone formation capacity of kidney cancer cells can be inhibited by silencing or knocking out the TPM3P9 protein, but the contrary is that the TPM3P9 protein is overexpressed, so that the TPM3P9 protein can be used for treating the kidney cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a TPM3P9 protein encoded by lncRNA and its application in tumor diagnosis and treatment. Background Art

[0002] Cancer, also known as malignant tumor, is caused by malignant cell proliferation and is invasive and metastatic. Common tumors include kidney cancer, gastric cancer, liver cancer, lung cancer, esophageal cancer, cervical cancer, breast cancer, colon cancer, intestinal cancer, etc. Among them, renal cell carcinoma (RCC) is a malignant tumor that occurs in renal tubular epithelial cells. It has a high mortality rate and is the most common malignant kidney tumor. Among them, renal clear cell carcinoma (ccRCC) is the most common pathological subtype. Early diagnosis of renal cancer is not obvious. Most renal cancer patients have developed to the middle and late stages at the time of diagnosis, which seriously affects the treatment effect. Due to the lack of effective early screening and diagnostic biomarkers, more than 15% of renal cancer patients have developed distant metastasis at the time of clinical diagnosis, and the 5-year survival rate of patients with metastatic renal cancer is less than 10%. At present, the main treatment for renal cancer is still radical surgery. Since renal cancer is insensitive to radiotherapy, gene targeted therapy provides a promising new direction for its treatment. Therefore, exploring new renal cancer biomarkers and therapeutic targets is the key to improving the early diagnosis rate and survival rate of renal cancer.

[0003] TPM3P9 (Homo sapiens tropomyosin 3pseudogene 9) is a long non-coding RNA with a length of 2932bp, but the relevant functions of TPM3P9 RNA have not been disclosed so far. Summary of the invention

[0004] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a TPM3P9 protein encoded by lncRNA.

[0005] Another object of the present invention is to provide an application of the TPM3P9 protein encoded by the lncRNA.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A TPM3P9 protein encoded by lncRNA, whose amino acid sequence is shown in SEQ ID NO.1.

[0008] The coding gene sequence of the TPM3P9 protein is any of the following sequences:

[0009] (a) The accession number in NCBI is NR_003148.3;

[0010] (b) As shown in SEQ ID NO.2.

[0011] Use of a reagent for detecting the expression level of the TPM3P9 protein in the preparation of a product for diagnosing or assisting in the diagnosis of tumors.

[0012] The tumor includes at least one of renal cancer, breast cancer, colorectal cancer, head and neck squamous cell carcinoma, uterine tumor, liver cancer and lung cancer; preferably renal cancer; more preferably renal clear cell carcinoma.

[0013] The products include test kits, test strips or chips.

[0014] Use of a reagent for detecting the expression level of the TPM3P9 protein in the preparation of a product for predicting the prognosis risk of renal cancer.

[0015] The products include test kits, test strips or chips.

[0016] The use of the TPM3P9 protein (as a target) in the preparation of a drug for treating renal cancer and / or inhibiting the proliferation of renal cancer cells.

[0017] The TPM3P9 is an important oncoprotein in the proliferation process of renal cancer. Targeting the TPM3P9 protein (knocking down the TPM3P9 protein) can effectively inhibit the proliferation and growth of renal cancer cells.

[0018] The treatment of renal cancer or the inhibition of renal cancer cell proliferation is achieved by silencing or knocking out the TPM3P9 gene (knocking down the TPM3P9 protein); wherein,

[0019] The siRNA sequence for silencing the TPM3P9 gene is preferably:

[0020] si-TPM3P9-sense: 5′-GGAAGAGAUGAAGAUUCUUTT-3′ (SEQ ID NO. 4);

[0021] si-TPM3P9-antisense: 5′-AAGAAUCUUCAUCUCUUCCTT-3′ (SEQ ID NO. 5);

[0022] The sgRNA sequence for targeted knockout of the TPM3P9 gene is preferably any of the following sequences:

[0023] sgRNA#1: 5′-CACCGCATTCAGGTCAAGCAAAGTCGTTT-3′ (SEQ ID NO. 6);

[0024] sgRNA#2: 5′-CACCGGCAGAGACCCATGCTGAGTGTTT-3′ (SEQ ID NO. 7).

[0025] The renal cancer includes renal clear cell carcinoma and the like.

[0026] The present invention combines immunoblotting and immunohistochemistry experiments to find that TPM3P9 protein is highly expressed in renal cancer and lowly expressed in (renal cancer) adjacent tissues; in addition, overexpression of TPM3P9 protein in renal cancer cells can promote the proliferation of renal cancer cells in mice in vivo and in vitro, and knocking down TPM3P9 significantly inhibits the proliferation of renal cancer cells. Therefore, the TPM3P9 protein provides a new diagnostic biomarker and therapeutic target for renal cancer.

[0027] Compared with the prior art, the present invention has the following advantages and effects:

[0028] 1. The present invention detected TPM3P9 protein in multiple human cell lines for the first time. TPM3P9 protein is a protein encoded by long non-coding RNA-TPM3P9 (lncRNA-TPM3P9).

[0029] 2. The present invention uses the immunoblotting method to detect the expression level of TPM3P9 protein in renal cancer and adjacent tissues, and finds that TPM3P9 protein is significantly differentially expressed in renal cancer tissues and adjacent tissues. The immunohistochemistry method is used to detect the expression level of TPM3P9 protein in 385 cases of renal cancer tissues, and finds that the expression level of TPM3P9 protein is correlated with the survival time of patients with renal clear cell carcinoma, indicating that TPM3P9 protein can be used as a highly efficient biomarker for the clinical diagnosis of renal clear cell carcinoma.

[0030] 3. The results of immunohistochemical analysis and survival analysis based on a large cohort of renal cancer and adjacent tissues in the present invention showed that TPM3P9 was highly expressed in renal clear cell carcinoma and lowly expressed in adjacent tissues, and its expression level was correlated with the survival time of renal cancer patients. The survival time of renal clear cell carcinoma patients with high expression of TPM3P9 was shorter, that is, high expression of TPM3P9 indicated a worse prognosis. These results indicate that TPM3P9, as an effective prognostic biomarker, can accurately predict the occurrence and development of renal clear cell carcinoma and has strong clinical application prospects.

[0031] 4. The present invention found through in vitro experiments that overexpression of TPM3P9 protein can promote the proliferation and cloning ability of renal cancer cells, and knocking out TPM3P9 protein can inhibit the proliferation and cloning ability of renal cancer cells; and in vivo experiments found that knocking out TPM3P9 can inhibit the subcutaneous growth of renal cancer cells in mice. Therefore, TPM3P9 can be used as a potential target for the treatment of renal cancer, and can be used in the treatment of renal cancer by targeting TPM3P9 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1It 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.

[0033] Figure 2 This is a diagram showing the expression of the new protein TPM3P9 in several common tumors and adjacent cancer tissues (in the figure, T: tumor tissue, N: adjacent cancer tissue).

[0034] Figure 3 The figure shows the expression of TPM3P9 protein in different renal cancer cell lines; A shows the expression of TPM3P9 in renal cancer cell lines ACHN and 786-O when overexpressed and knocked down; B shows the expression of TPM3P9 in renal tubular epithelial cells HK-2 and 9 renal cancer cell lines.

[0035] Figure 4 Figure 2 is the expression of TPM3P9 protein in renal cancer and adjacent tissues; A is the expression and statistical graph of TPM3P9 in 16 human renal cancer tissues and adjacent tissues (in the figure, T: renal cancer tissue, N: adjacent tissue); B is the statistical result of TPM3P9 protein expression in 16 human renal cancer tissues and adjacent tissues; C is the expression statistics of lncRNA-TPM3P9 in 72 pairs of renal cancer and adjacent tissues in the TCGA dataset; D is the expression statistics of TPM3P9 in 81 pairs of renal cancer and adjacent tissues in the CPTAC dataset.

[0036] Figure 5 The figure is a curve diagram showing the relationship between the expression of TPM3P9 protein and the survival time of patients with renal cancer; A is the staining diagram of high and low expression of TPM3P9; B is the overall survival curve (OS); and C is the recurrence-free survival curve (RFS).

[0037] Figure 6Figure 1 is the effect of TPM3P9 protein on the proliferation of renal cancer cells; A is the lncRNA in RNA form that constructs TPM3P9 protein and mutates the start codon and stop codon; B is the immunoblotting method that proves the successful overexpression of TPM3P9 protein in renal cancer cells; C is the qRT-PCR method that proves the successful overexpression of TPM3P9 and lncRNA-TPM3P9 in renal cancer cells; D is the ability of overexpression of TPM3P9 protein to promote the growth of renal cancer cells (CCK-8 experiment); E is the ability of overexpression of TPM3P9 protein to promote the cloning of renal cancer cells; F is the ability of overexpression of TPM3P9 to promote renal cancer cells Proliferation (EdU experiment); G is the successful knockdown of TPM3P9 protein in renal cancer cells; H is the ability of silencing TPM3P9 protein to inhibit the growth of renal cancer cells (CCK-8 experiment); I is the ability of knocking down TPM3P9 protein to inhibit the cloning of renal cancer cells; J is the inhibition of proliferation of renal cancer cells by knocking down TPM3P9 protein (EdU experiment); K is the growth curve of renal cancer cells in mice inhibited by knocking down TPM3P9 protein; L is the inhibition of renal cancer cell growth in mice by knocking down TPM3P9 protein; M is the weight of subcutaneous tumors in mice inhibited by knocking down TPM3P9 protein; N is the weight change of mice during the in vivo experiment. DETAILED DESCRIPTION

[0038] 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.

[0039] Example 1 Identification of TPM3P9 protein

[0040] 1. The present invention first 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,

[0041] The TPM3P9 protein sequence (SEQ ID NO.1) is as follows:

[0042] MDEQIRLMDQNLKCLSAAEEKYSQKEDKCEEEMKILTDNLKEAETHAELAERSVAKL EKTIDDLEDKLKCTKEEHLCTQRMLDQTLLDLNEM;

[0043] The TPM3P9 gene sequence (SEQ ID NO.2) is as follows:

[0044] ATGGATGAGCAGATCAGACTGATGGACCAGAACCTGAAGTGTCTGAGTGCAGCTGAAGAAAAGTACTCTCAAAAAGAAGACAAATGTGAGGAAGAGATGAAGATTCTTACTGATAATCTCAAGGAGGCAGAGACCCATGC TGAGTTGGCTGAGAGATCAGTAGCCAAGCTGGAAAAGACAATTGATGACTTGGAAGATAAACTGAAATGCACCAAAGAGGAACACCTCTGTACACAAAGGATGCTGGACCAGACTTTGCTTGACCTGAATGAGATGTAG.

[0045] 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.

[0046] 2. This experiment further provides protein evidence for TPM3P9 identification by mass spectrometry. The specific experimental steps are as follows:

[0047] (1) Protein enzymatic hydrolysis: Renal cancer cell lines ACHN and 786-O (purchased from ATCC) were collected and lysed to obtain a 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 placed at 37°C for 16 hours. Finally, the peptides obtained by enzymatic hydrolysis were freeze-dried in a vacuum dryer.

[0048] (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.

[0049] (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.

[0050] 3. Preparation of anti-TPM3P9 polyclonal antibody

[0051] (1) The gene coding sequence of TPM3P9 protein (SEQ ID NO. 2) and pGEX-4T-1 plasmid (commonly available in the market) were digested with restriction endonucleases Xho I and BamH I, and then ligated with T4 ligase to obtain the recombinant plasmid pGEX-4T-1-TPM3P9;

[0052] (2) The recombinant plasmid pGEX-4T-1-TPM3P9 was transformed into E. coli BL21 and cultured in a 37°C constant temperature shaker for about 2 hours. 600 = 0.6, add 0.5mM IPTG (isopropyl-β-D-thiogalactoside) to induce for 2 to 6 hours, collect the precipitate of the bacteria by centrifugation at low temperature, and resuspend and wash 3 times with PBS. Repeat freezing and thawing 3 times, then use ultrasound to break the bacteria, centrifuge at 4℃, 12000g for 30 minutes, and retain the supernatant. Add 1mL of GST-tagPurification Resin to the empty column tube of the affinity chromatography column and pass it twice with PBS. 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 all the bacterial protein supernatant is filtered, add PBS and filter 4 times to wash away the impurities. Prepare the eluent, a 50mM Tris-HCl solution with a final concentration of 10mM GSH (glutathione), pH 8.0. Add the eluent and collect the liquid that flows out. The above liquid was concentrated in a 10kDa ultrafiltration tube and finally washed twice with PBS. The purified fusion protein was digested with thrombin (4°C, digested for 12 hours, 10 units of enzyme digested 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.

[0053] (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;

[0054] (4) Anti-TPM3P9 antibodies were purified from Japanese white rabbit serum (commissioned by Wuhan Proteintech Group).

[0055] Example 2 Screening for TPM3P9 as a biomarker for the diagnosis of renal cell carcinoma

[0056] 1. Analyze multiple types of tumor and adjacent tumor data in the CPTAC database (Clinical Proteomic Tumor Analysis Consortium, https: / / proteomics.cancer.gov / programs / cptac), including renal cancer, breast cancer, colorectal cancer, head and neck squamous cell carcinoma, uterine tumor, liver cancer and lung cancer. The analysis results are as follows Figure 2 As shown, the new protein TPM3P9 was found to be differentially expressed in several common tumors and adjacent cancer tissues. The expression of TPM3P9 in renal cancer tissue was significantly higher than that in adjacent cancer tissues. The expression of TPM3P9 in breast cancer, colorectal cancer, head and neck squamous cell carcinoma, uterine tumor and liver cancer tissues was also significantly higher than that in adjacent cancer tissues. However, the expression of TPM3P9 in lung cancer tissue was significantly lower than that in adjacent cancer tissues.

[0057] 2. The present invention selects renal cancer tissue / cell samples for further verification, and the specific steps are as follows:

[0058] (1) TPM3P9 was overexpressed or knocked down in renal cancer cell lines ACHN and 786-O, and the overexpression or knockdown of TPM3P9 was verified using the TPM3P9 antibody prepared as described above.

[0059] 1) Construction of TPM3P9 plasmid: The recombinant plasmid pLVX-Puro-TPM3P9-flag was constructed on the backbone of pLVX-Puro Vector (Addgene). The pLVX-Puro empty plasmid (pLVX-Puro Vector) was used as a control. The gene sequence of TPM3P9 was constructed to be consistent with the above-mentioned SEQ ID NO.2. The pLVX-Puro plasmid and the synthetic TPM3P9-flag fusion sequence (commissioned by Addgene Biotech) were double-digested with restriction endonucleases EcoR I and BamH I, and the T4 DNA enzyme was connected to construct the corresponding recombinant plasmid pLVX-Puro-TPM3P9-flag.

[0060] TPM3P9-flag sequence (SEQ ID NO.3):

[0061] GAATTCGCCACCGAACGAGCTGAGCTGGCAGAGTCCCGTTGCTGAGAGATGGATGAGCAGATCAGACTGATGGACCAGAACCTGAAGTGTCTGAGTGCAGCTGAAGAAAAGTACTCTCAAAAAGAAGACAAATGTGAGGAAGAGATGAAGATTCTTACTGATAATCTCAAGGAGGCAGAGACCCATGCTGAGTTGGCTGA GAGATCAGTAGCCAAGCTGGAAAAGACAATTGATGACTTGGAAGATAAACTGAAATGCACCAAAGAGGAACACCTCTGTACACAAAGGATGCTGGACCAGACTTTGCTTGACCTGAATGAGATGGACTACAAAGACATGACGGTGATTATAAAAGATCATGACATCGATTACAAGGATGACGATGACAAGTAGGGATCC.

[0062] 2) Entrust Aiki Biotech to synthesize TPM3P9 interfering RNA (siTPM3P9) and control interfering RNA (siNC), wherein the sequence of TPM3P9 interfering RNA is as follows (5′-3′): si-TPM3P9-sense:

[0063] GGAAGAGAUGAAGAUUCUUTT (SEQ ID NO. 4); si-TPM3P9-antisense: AAGAAUCUUCAUCUCUUCCTT (SEQ ID NO. 5).

[0064] 3) Transient overexpression of TPM3P9: ACHN or 786-O cells were plated in a six-well plate. When the cells grew to a density of about 30-50%, 1 μg of pLVX-Puro empty plasmid and pLVX-TPM3P9-flag recombinant plasmid were taken respectively, and transiently transfected with Lipo3000 4 μL / tube; 48 hours after transfection, the cells were collected and the overexpression effect was verified by immunoblotting. The experiment was repeated three times.

[0065] 4) Transient knockdown of TPM3P9: ACHN or 786-O cells were plated in a six-well plate. 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, the cells were collected and the knockdown effect was verified by immunoblotting. The experiment was repeated three times.

[0066] The results are as follows Figure 3 As shown in A, the results of immunoblotting showed that the TPM3P9 antibody prepared above could successfully detect the overexpression and knockdown of TPM3P9 in renal cancer cell lines ACHN and 786-O.

[0067] (2) Immunoblotting was performed to detect the expression of TPM3P9 in 9 renal cancer cell lines (ACHN, Caki-1, Caki-2, OR-SC-2, SN12C, UMRC-6, A498, 769-P, 786-O) (all purchased from ATCC) and renal tubular epithelial cells HK-2 (purchased from ATCC). The specific steps are as follows: For renal cancer cell lines, pour out the cell culture medium and wash three times with PBS buffer. Add lysis buffer (containing phenylmethylsulfonyl fluoride PMSF) and place on ice for lysis for 30 minutes. Centrifuge at 4°C, 12000rpm, and transfer the obtained protein supernatant to a 1.5mL centrifuge tube. After measuring the protein concentration, take a certain amount of protein and sample buffer to prepare the sample, and place it in a 100°C water bath for heating and denaturation. Then perform protein electrophoresis, transfer to the membrane and block. Finally, the primary antibody was incubated with anti-TPM3P9 antibody (prepared in Example 1), the primary antibody was recovered, washed with TBST, and then the secondary antibody was added and incubated at room temperature, and then developed after washing with TBST.

[0068] The results are as follows Figure 3 As shown in B, the results showed that TPM3P9 was ubiquitously expressed in a variety of renal cancer cell lines, and the expression of TPM3P9 in renal cancer cell lines was significantly higher than that in normal renal cells (renal tubular epithelial cells HK-2).

[0069] (2) Next, 16 pairs of renal cancer tumor samples and adjacent tissue samples (from Sun Yat-sen University Cancer Center) were subjected to immunoblotting (the detection method was the same as in step (1) above), and the difference in TPM3P9 protein expression in tumor samples and adjacent tissue samples was statistically analyzed.

[0070] The results are as follows Figure 4 A and Figure 4 As shown in B, the results of immunoblotting showed that TPM3P9 was significantly overexpressed specifically in renal clear cell carcinoma compared with adjacent adjacent tissues (P=0.0019).

[0071] (3) We downloaded RNA sequencing data of 72 cases of renal cancer and their adjacent adjacent cells from The Cancer Genome Atlas (https: / / cancergenome.nih.gov / ) and mass spectrometry data of 81 pairs of renal cancer and their adjacent adjacent cells from the CPTAC database, and performed statistical analysis on the RNA expression level and protein expression level of TPM3P9 in renal cancer and their adjacent adjacent cells.

[0072] The results are as follows Figure 4 C and Figure 4 As shown in D, the statistical results showed that the RNA (TCGA) and protein (CPTAC) of TPM3P9 were significantly overexpressed in renal cancer tissues compared with adjacent adjacent tissues.

[0073] (4) A tissue chip containing clinical samples of 385 patients with renal cancer (the tissue chip was obtained from the Cancer Center of Sun Yat-sen University) was selected for immunohistochemical detection of TPM3P9 protein and survival correlation analysis. The specific steps are as follows:

[0074] 1) Staining: The paraffin sections corresponding to the above tissue chip were incubated with anti-TPM3P9 polyclonal antibody (prepared in Example 1) 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.).

[0075] 2) Survival analysis was performed on the patients based on the immunohistochemistry results: the patients were divided into two groups according to the total TPM3P9 staining score, the TPM3P9 high expression group (194 cases) and the low expression group (191 cases), and the survival curve was fitted based on the corresponding survival time (OS and RFS).

[0076] The results are as follows Figure 5 As shown: Figure 5 The immunohistochemical results of A show the staining of high and low expression of TPM3P9 in renal cancer tissues. In addition, according to the expression of TPM3P9, renal cancer patients were divided into two groups: TPM3P9 high expression group and low expression group. The overall survival curve (OS) was fitted based on the corresponding survival time. Figure 5 B shows the recurrence-free survival (RFS) curve. Figure 5 As shown in Figure C, high expression of TPM3P9 indicates a worse prognosis for patients with renal cancer, indicating that there is a significant negative correlation between the expression level of TPM3P9 and the survival time of patients.

[0077] The above results indicate that TPM3P9 can be used as an effective biomarker for the diagnosis of renal cancer and has application value in the clinical diagnosis of renal cancer.

[0078] Example 3 TPM3P9 can be used as a potential target for the treatment of renal cell carcinoma

[0079] 1. Experiment on TPM3P9 protein promoting renal cancer cell clone formation and cell growth

[0080] 1. ACHN and 786-O cells (both purchased from ATCC) that stably overexpress TPM3P9 protein and lncRNA in RNA form with mutated start and stop codons were constructed, and verified by Western blotting and qRT-PCR experiments. The specific steps are as follows:

[0081] (1) Plasmid construction: The recombinant plasmids pLVX-Puro-TPM3P9-flag and pLVX-Puro-Mut were constructed on the backbone of pLVX-Puro Vector (Addgene) (where the Mut group represents a vector lacking the ATG start codon, which cannot express protein but can express RNA) Figure 6 A). Use pLVX-Puro empty vector as control. Specifically:

[0082] 1) The gene sequence of TPM3P9 was consistent with the above SEQ ID NO.2. The lncRNA-TPM3P9 was constructed by mutating the start codon ATG to GGA and the stop codon from TGA to GCT based on the SEQ ID NO.2 sequence. The full sequence was synthesized into a fusion sequence of TPM3P9-flag (SEQ ID NO.3) and lncRNA-TPM3P9-flag (both were commissioned to be synthesized by Aiki Biotech).

[0083] 2) The pLVX-Puro plasmid and the fusion sequences of the synthesized TPM3P9-flag and lncRNA-TPM3P9-flag were double-digested with restriction endonucleases EcoR I and BamH I, and ligated with T4 DNA enzyme to construct the corresponding recombinant plasmids pLVX-Puro-TPM3P9-flag and pLVX-Puro-Mut.

[0084] (2) Construction of stable cell lines: 293T cells were plated in six-well plates. When the cell density reached about 80%, 1 μg of pLVX-Puro empty vector plasmid, pLVX-Puro-TPM3P9-flag, and pLVX-Puro-Mut recombinant plasmids and lentiviral packaging plasmids psPAX2 and pMD2.G (Addgene) were taken and transfected into six-well plates using 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 cells ACHN and 786-O. After 48 hours of virus infection, the medium was changed, and then 2 μg / mL of puromycin was added for screening of ACHN and 786-O overexpression positive cells. Puromycin was withdrawn when there was no significant change in the cell number.

[0085] (3) Western blotting experiment to detect the overexpression effect: collect cells and verify the overexpression effect through Western blotting experiment and qRT-PCR experiment; freeze the successfully constructed cell line and store it in a -80℃ liquid nitrogen tank.

[0086] The results are as follows Figure 6 As shown in B and 6C: Immunoblotting results showed that the use of the TPM3P9 antibody prepared above was able to successfully detect the overexpression of TPM3P9 protein in ACHN and 786-O cells transfected with TPM3P9 wild-type ORF instead of Mut; qRT-PCR results showed that TPM3P9 mRNA expression was upregulated in ACHN and 786-O cells transfected with TPM3P9 wild-type ORF or mutant Mut.

[0087] 2. Stable overexpression of TPM3P9 protein promoted tumor proliferation of renal cancer cells in vitro. The specific experimental method is as follows:

[0088] (1) CCK-8 experiment: The above-constructed stably overexpressing TPM3P9 protein and the control ACHN and 786-O cells were trypsinized 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% (v / v) 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. The experiment was set up with three replicates.

[0089] (2) Clone formation experiment: The above-constructed stably overexpressing TPM3P9 protein and the control ACHN and 786-O cells were trypsinized to prepare single-cell suspensions, and the cells were counted and inoculated into 6-well plates at a density of 1,000 cells per well. They were cultured at 37°C for 10 to 14 days to determine the cell cloning ability. After 10 days of culture, the original culture medium was discarded, the cells were washed twice with PBS, fixed with methanol, and stained with 0.5% (w / v) crystal violet solution. Then, the cell clones were photographed and counted using ImageJ software. The experiment was repeated three times.

[0090] (3) EdU experiment: The above-constructed stably overexpressing TPM3P9 protein and control ACHN and 786-O cells were cultured in 12-well plates and treated with 100 μL of culture medium containing 20 μM EdU. After incubation for 2 hours at 37°C and 5% CO2, the cells were fixed with 4% paraformaldehyde for 30 minutes and incubated with 0.5% Triton-X-100 in PBS for 20 minutes. The cell nuclei were stained with DAPI. Five randomly selected field images of each group were collected using a fluorescence microscope and analyzed using ImageJ software to calculate the proliferation rate. The experiment was repeated three times.

[0091] The results are as follows Figure 6 D. Figure 6 As shown in E and 6F: CCK-8, clone formation and EdU experiments all showed that stable overexpression of TPM3P9 protein can significantly promote the proliferation and clone formation ability of renal cancer cells.

[0092] 3. Construct ACHN and 786-O cells with stable TPM3P9 knockout and knockout control ACHN and 786-O cells, and verify them by Western blotting. The specific steps are as follows:

[0093] (1) Plasmid construction: The lentiCRISPR v2 (Addgene) plasmid was cut with restriction endonucleases Age I and EcoR I, and then the sgRNA #1 fragment (CACCGCATTCAGGTCAAGCAAAGTCGTTT) (SEQ ID NO. 6) and the sgRNA #2 fragment (CACCGGCAGAGACCCATGCTGAGTGTTT) (SEQ ID NO. 7) (5′-3′) (commissioned to Aiki Biotechnology Co., Ltd. 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.

[0094] (2) Construction of stable cell lines: 293T cells were plated in six-well plates. When the cell density reached about 80%, 1 μg of the control group sg NC (Addgene) and 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 cells ACHN and 786-O. 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 knockout positive cells. The puromycin was withdrawn after there was no significant change in the cell number.

[0095] (3) Western blotting experiment to detect the knockdown effect: collect cells and verify the knockout effect by Western blotting experiment; freeze the successfully constructed cell line and store it in a -80℃ liquid nitrogen tank.

[0096] The results are as follows Figure 6 G: Immunoblotting results showed that TPM3P9 protein was successfully knocked down in ACHN and 786-O cells.

[0097] 4. Stable knockdown of TPM3P9 protein inhibited tumor proliferation of renal cancer cells in vitro. The specific experimental methods included CCK-8, clone formation, and EdU experimental steps, which were the same as those in step 2 above.

[0098] The results are as follows Figure 6 H. Figure 6 I and Figure 6 J shows that CCK-8, clone formation and EdU experiments all indicate that ACHN and 786-O cells with stable knockdown of TPM3P9 protein can significantly inhibit the proliferation and clone formation ability of renal cancer cells.

[0099] 2. Experimental study on TPM3P9 protein promoting tumor proliferation of renal cancer cells in mice

[0100] 1. Experiment on in vivo tumor proliferation of mice with stable knockout of TPM3P9 protein to inhibit renal cancer cells. The specific experimental methods are as follows:

[0101] 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):

[0102] (1) Each mouse was subcutaneously inoculated with 5×10 6ACHN 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;

[0103] (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;

[0104] (3) Observe the growth status of mice and remove the tumors at 30 days for follow-up studies.

[0105] The results are as follows Figure 6 As shown in K~6N, the mouse subcutaneous tumor formation experiment showed that knocking down TPM3P9 could inhibit the subcutaneous growth of renal cancer cells in mice ( Figure 6 K and Figure 6 L), knockdown of TPM3P9 significantly reduced the size and weight of subcutaneous tumors in mice ( Figure 6 M), while there was no significant difference in the body weight of mice in each treatment group during the experiment ( Figure 6 N). In vivo experiments have shown that TPM3P9 can promote the proliferation and growth of renal cancer cells. TPM3P9 is a biomarker related to renal cancer proliferation. Targeting TPM3P9 can provide a therapeutic target for anti-renal cancer proliferation and has clinical application value for renal cancer.

[0106] 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. A TPM3P9 protein encoded by lncRNA, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. The coding gene sequence of the TPM3P9 protein according to claim 1, 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.

3. Use of a reagent for detecting the expression level of the TPM3P9 protein according to claim 1 in the preparation of a product for diagnosing or assisting in the diagnosis of tumors.

4. The use according to claim 3, characterized in that: The tumor includes at least one of renal cancer, breast cancer, colorectal cancer, head and neck squamous cell carcinoma, uterine tumor, liver cancer and lung cancer.

5. The use according to claim 4, characterized in that: The tumor is renal cancer.

6. Use of a reagent for detecting the expression level of the TPM3P9 protein according to claim 1 in the preparation of a product for predicting the prognosis risk of renal cancer.

7. The use according to any one of claims 3 to 6, characterized in that: The products include test kits, test strips or chips.

8. Use of the TPM3P9 protein according to claim 1 in the preparation of a drug for treating renal cancer and / or inhibiting the proliferation of renal cancer cells.

9. The use according to claim 8, characterized in that: The treatment of renal cancer or the inhibition of renal cancer cell proliferation is achieved by silencing or knocking out the TPM3P9 gene; wherein, The siRNA sequences used to silence the TPM3P9 gene are as follows: 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′.

10. The use according to claim 8, characterized in that: The renal cancer includes renal clear cell carcinoma.

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