Application of therapeutic target to products for inhibiting transfer and proliferation of kidney cancer cells

Abstract

The invention discloses an application of a treatment target in a product for inhibiting transfer and proliferation of kidney cancer cells. The nucleotide sequence of the C8orf76 treatment target is as shown in SEQ ID NO. 1; the expression protein of the C8orf76 therapeutic target spot is a C8orf76 protein; the amino acid sequence of the C8orf76 protein is as shown in SEQ ID NO. 2. The invention discloses C8orf76 with a nucleotide sequence as shown in SEQ ID NO.1 and C8orf76 protein with an amino acid sequence as shown in SEQ ID NO.2, and discloses application of the C8orf76 protein in preparation of products for inhibiting transfer and proliferation of kidney cancer cells, and the C8orf76 protein is a new target for diagnosis and gene therapy of ccRCC.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of a therapeutic target in a product for inhibiting the metastasis and proliferation of renal cancer cells. Background Art

[0002] Clear cell renal cell carcinoma (ccRCC) is the most common malignant renal tumor and is prone to metastasis. It is difficult to detect in the early stages of the disease. Nearly half of patients are already in the advanced stage when diagnosed. At this time, the tumor has already invaded locally and metastasized to distant sites. Simple surgical treatment cannot benefit the survival of advanced patients. In addition, the tumor is prone to develop resistance to existing immune checkpoint inhibitors and tyrosine kinase inhibitors, which seriously affects the patient's prognosis and quality of life. Therefore, finding tumor-specific therapeutic targets has been a hot topic in renal cancer research in recent years and is also the key to solving clinical difficulties.

[0003] Tumor diagnosis and treatment has entered the era of molecular classification. Existing molecular experimental techniques can achieve the purpose of diagnosis and treatment by detecting tumor-specific markers. In the field of the urinary system, for example, prostate cancer specifically expresses androgen receptors, so endocrine therapy drugs target blocking the receptor signaling pathway to achieve the purpose of inhibiting tumor growth. When the tumor differentiates into a neuroendocrine type, the expression level of androgen receptors will decrease significantly, and endocrine therapy is no longer the first choice. For renal cancer, there is still a lack of relevant specific molecules for diagnosis and treatment. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a therapeutic target in a product for inhibiting the metastasis and proliferation of renal cancer cells, so as to solve the technical problem of the lack of a therapeutic target for inhibiting the metastasis and proliferation of renal cancer cells in the prior art.

[0005] In order to solve the above technical problems, the present invention specifically provides the following technical solutions:

[0006] The present invention provides an application of a therapeutic target in a product for inhibiting the metastasis and proliferation of renal cancer cells, wherein the nucleotide sequence of the C8orf76 therapeutic target is shown in SEQ ID NO.1;

[0007] The expression protein of the C8orf76 therapeutic target is C8orf76 protein;

[0008] The amino acid sequence of the C8orf76 protein is shown in SEQ ID NO.2.

[0009] As a preferred embodiment of the present invention, the product is a kit;

[0010] The kit contains reagents for knocking out or silencing the C8orf76 therapeutic target;

[0011] The reagent includes siRNA, shRNA or miRNA;

[0012] The functional sequence of the shRNA is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0013] As a preferred embodiment of the present invention, the product is an anti-kidney cancer drug;

[0014] The anti-renal cancer drug comprises a pharmaceutically acceptable carrier and a C8orf76 protein inhibitor.

[0015] As a preferred embodiment of the present invention, the C8orf76 protein inhibitor is a 1,2,3,6-Tetragalloy1lucose inhibitor, which can bind to the C8orf76 protein and inhibit its function.

[0016] As a preferred embodiment of the present invention, the C8orf76 protein inhibitor is any one or more of a protease that degrades C8orf76 products, a nuclease that degrades C8orf76 products, or a regulator that reduces C8orf76 products.

[0017] As a preferred embodiment of the present invention, the C8orf76 product reducing regulator is a C8orf76 antibody.

[0018] As a preferred embodiment of the present invention, the carrier is any one or more of lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, ethylene glycol, water, saline or glucose aqueous solution.

[0019] As a preferred embodiment of the present invention, the anti-kidney cancer drug further comprises an antioxidant;

[0020] Wherein, the antioxidant is any one of ascorbic acid, sodium sulfite or sodium bisulfate.

[0021] As a preferred embodiment of the present invention, the anti-kidney cancer drug further comprises an excipient;

[0022] Wherein, the excipient is any one of corn starch, wheat starch, methyl cellulose, sodium carboxymethyl cellulose or gelatin.

[0023] As a preferred embodiment of the present invention, the administration routes of the anti-renal cancer drug include oral, intravenous, parenteral, intramuscular, subcutaneous, intraperitoneal, intranasal, rectal or local administration.

[0024] As a preferred embodiment of the present invention, the dosage of the C8orf76 protein inhibitor is 0.01 μg to 1000 mg / day per patient.

[0025] As a preferred embodiment of the present invention, the renal cancer cell is ccRCC.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention discloses an application of C8orf76 mRNA as a therapeutic target in the preparation of a drug for treating renal cancer. After C8orf76 is knocked out or silenced by using shRNA (see SEQ ID NO.3 and SEQ ID NO.4), the proliferation and migration abilities of 786-O and 769-P cells are significantly inhibited after the C8orf76 gene is knocked down by lentivirus, and tumor growth is significantly inhibited at the animal level.

[0028] This invention proposes and verifies for the first time that C8orf76 protein is a new target for the diagnosis and gene therapy of ccRCC, which is of great significance for the screening of new anti-renal cancer drugs and provides a new idea for the diagnosis and treatment of renal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0030] Figure 1 Provided for the present invention is a graph showing the difference in expression between (unpaired) renal cancer tumors and non-tumor tissues of the C8orf76 gene in TCGA-KIRC database in Verification Example 1;

[0031] Figure 2 Provided for the present invention is a graph showing the difference in expression between (paired) renal cancer and adjacent tissues of the C8orf76 gene in TCGA-KIRC database in Verification Example 1;

[0032] Figure 3 Provided is a graph showing the results of the area under the receiver operating characteristic (ROC) curve (AUC) of C8orf76 in the diagnosis of renal cancer in Verification Example 1 of the present invention;

[0033] Figure 4 Provided for the present invention is a graph showing the effect of different expression levels of C8orf76 on the patient's tumor-specific survival (DSS) in Verification Example 1;

[0034] Figure 5 Provided for the present invention is a graph showing the effect of different expression levels of C8orf76 on the progression-free survival (PFI) of patients in Verification Example 1;

[0035] Figure 6 Provided for the present invention is a graph showing the effect of different expression levels of C8orf76 on the overall survival (OS) of patients in Verification Example 1;

[0036] Figure 7 The present invention provides an immunoblot electrophoresis diagram of C8orf76 after tissue protein extraction of renal cancer and adjacent tissue specimens obtained in the research center in Verification Example 2;

[0037] Figure 8 Provided for the present invention is a qPCR result diagram of the difference in C8orf76 expression at the mRNA level between the normal renal tubular epithelial cell line (HK-2) and the ccRCC cell line in Verification Example 3;

[0038] Fig. 9 Provided for the present invention is an immunoblot electrophoresis diagram of the difference in C8orf76 expression at the protein level between the normal renal tubular epithelial cell line (HK-2) and the ccRCC cell line in Verification Example 4;

[0039] Fig.10 The present invention provides an efficiency diagram of mRNA and protein levels of the knockdown group and the control group after knocking down C8orf76 with a lentivirus in the ccRCC cell line 786-O in Example 1;

[0040] Fig.11 The present invention provides an efficiency diagram of mRNA and protein levels of the knockdown group and the control group after knocking down C8orf76 in the ccRCC cell line 769-P using a lentivirus in Example 1;

[0041] Fig.12 The present invention provides a graph showing the effect of stable knockdown of C8orf76 by lentivirus on tumor proliferation in the ccRCC cell line 786-O in Example 1;

[0042] Fig.13 The present invention provides a graph showing the effect of stable knockdown of C8orf76 by lentivirus on tumor proliferation in the ccRCC cell line 769-P in Example 1;

[0043] Fig.14 The present invention provides a graph showing the effect of stable knockdown of C8orf76 by lentivirus on tumor metastasis ability in the ccRCC cell line 786-O in Example 1;

[0044] Fig.15 The present invention provides a graph showing the effect of stable knockdown of C8orf76 by lentivirus on tumor metastasis ability in the ccRCC cell line 769-P in Example 1;

[0045] Fig.16The present invention provides a graph showing the effect of stable knockdown of C8orf76 by lentivirus on tumor growth ability after constructing a ccRCC subcutaneous transplant tumor model at the animal level in Example 1;

[0046] Fig.17 Provides a cartoon diagram of the binding of 1,2,3,6-Tetragalloylglucose and C8orf76 in Example 2 of the present invention;

[0047] Fig.18 Provides a surface diagram of the binding of 1,2,3,6-Tetragalloylglucose and C8orf76 in Example 2 of the present invention;

[0048] Fig.19 Provides a 2D diagram of the binding mode of 1,2,3,6-Tetragalloylglucose and C8orf76 in Example 2 of the present invention;

[0049] Fig. 20 Provides a 3D map of the binding of 1,2,3,6-Tetragalloylglucose and C8orf76 in Example 2 of the present invention;

[0050] Fig.21 The present invention provides a tumor proliferation inhibition diagram of the 1,2,3,6-Tetragalloylglucose inhibitor on the inhibition of C8of76 protein function in Example 2. DETAILED DESCRIPTION

[0051] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] The present invention discloses the application of C8orf76 mRNA in ccRCC. Chromosome 8 open reading frame 76 (C8orf76) is a nuclear protein encoding gene. Existing studies have shown that C8orf76 can affect the occurrence and development of liver cancer, gastric cancer and breast cancer. However, the role of C8orf76 in ccRCC is still unclear, and there are few studies on this gene. In-depth exploration of its influence on the diagnosis and treatment of ccRCC is of great value.

[0053] The C8orf76 protein of the present invention may be human C8orf76, and more specifically, the nucleotide sequence of C8orf76 may be or include the sequence of NCBI reference sequence: NM_032847.3.

[0054] The nucleotide sequence of the C8orf76 therapeutic target is shown in SEQ ID NO.1.

[0055] SEQ ID NO.1:

[0056] ATGGATTCCGGGTGCTGGTTGTTCGGCGGCGAGTTCGAGGACTCGGTGTTCGAGGAGAGGCCGGAGCGGCGGTCAGGACCGCCCGCGTCCTACTGCGCCAAGCTCTGCGAGCCGCAGTGGTTTTATGAAGAAACAGAAAGCAGTGATGATGTTGAAGTGCTGACTCTCAAGAAATTCAAAGGAGACCTGGCCTACAGACGACAAGAGTATCAGAAAGCACTGCAGGAGTATTCCAGTATCTCTGAAAAATTGTCATCAACCAATTTTGCCATGAAAAGGGATGTCCAGGAAGGTCAGGCTCGGTGTCTGGCTCACCTGGGTAGGCATATGGAGGCGCTGGAGATTGCTGCAAACTTGGAAAATAAAGCAACCAACACAGACCATTTAACCACGGTACTCTACCTCCAGCTTGCTATTTGTTCAAGTTTGCAGAACTTGGAGAAAACAATTTTCTGCCTGCAGAAACTGATTTCTTTGCATCCTTTTAATCCTTGGAACTGGGGCAAATTGGCAGAGGCTTACCTGAATCTGGGGCCAGCTCTTTCAGCAGCACTTGCGTCATCTCAGAAACAGCACAGTTTCACCTCAAGTGACAAAACTATCAAATCCTTCTTTCCACACTCAGGAAAAGACTGTCTTTTGTGTTTTCCTGAAACCTTGCCTGAGAGCTCTTTATTTTCTGTGGAAGCGAATAGCAGTAATAGCCAGAAAAATGAGAAAGCTCTGACAAATATCCAAAACTGTATGGCAGAAAAGAGAGAAACAGTGTTGATAGAGACTCAGCTGAAAGCATGTGCCTCTTTTATACGAACCAGGCTTCTGCTTCAGTTTACCCAACCTCAGCAAACATCGTTTGCTTTGGAGAGGAACTTAAGGACTCAGCAGGAAATTGAAGATAAAATGAAAGGGTTCAGCTTCAAAGAAGACACTTTGCTGTTGATAGCTGAGGTTATGGGAGAAGATATCCCAGAAAAAATAAAAGATGAAGTTCACCCAGAGGTGAAGTGTGTTGGCTCCGTAGCCCTGACTGCCTTGGTGACTGTATCCTCAGAAGAATTTGAAGACAAGTGGTTCAGAAAGATCAAAGACCATTTCTGTCCATTTGAAAATCAGTTCCATACAGAGATACAAATCTTGGCTTAG

[0057] The expression protein of the C8orf76 therapeutic target is the C8orf76 protein, and the amino acid sequence of the C8orf76 protein can be or include but is not limited to an amino acid sequence that has at least 80%, 85%, 90% or 95% identity with the NCBI reference sequence: NP_116236.1 sequence, as well as an amino acid sequence that has the characteristics or functions of C8orf76.

[0058] The amino acid sequence of the C8orf76 protein is shown in SEQ ID NO.2.

[0059] SEQ ID NO.2:

[0060] MDSGCWLFGGEFEDSVFEERPERRSGPPASYCAKLCEPQWFYEETESSDDVEVLTLKKFKGDLAYRRQEYQKALQEYSSISEKLSSTNFAMKRDVQEGQARCLAHLGRHMEALEIAANLENKATNTDHLTTVLYLQLAICSSLQNLEKTIFCLQKLISLHPFNPWNWGKLAEAYLNLGPALSAALASSQK QHSFTSSDKTIKSFFPHSGKDCLLCFPETLPESSLFSVEANSSNSQKNEKALTNIQNCMAEKRETVLIETQLKACASFIRTRLLLQFTQPQQTSFALERNLRTQQEIEDKMKGFSFKEDTLLLIAEVMGEDIPEKIKDEVHPEVKCVGSVALTALVTVSSEEFEDKWFRKIKDHFCPFENQFHTEIQILA

[0061] Among the applicable products, there may be a kit containing a regulator for reducing the expression of C8orf76, wherein the regulator is a reagent for knocking out or silencing C8orf76. Further preferably, the reagent for knocking out or silencing C8orf76 comprises siRNA, shRNA or miRNA.

[0062] Among the products that can be applied, they can also exist in the form of drugs and can be administered through conventional drug administration methods such as injection. For example, silencing C8orf76 can be achieved by introducing siRNA of a specific sequence into the body, similar to the introduction method of the new crown mRNA vaccine.

[0063] In a preferred embodiment, the siRNA refers to a short double-stranded RNA that can induce RNA interference by inducing degradation of certain mRNAs. The siRNA includes a sense RNA chain having a sequence homologous to the mRNA of the target gene and an antisense RNA chain having a sequence complementary to the mRNA. The siRNA can inhibit the expression of the target gene and can be used for gene knockdown and gene therapy.

[0064] In a preferred embodiment, the shRNA (short hairpin RNA) is a single-stranded RNA, which includes a stem portion and a loop portion that form a double-stranded portion through hydrogen bonds, and is processed and converted into siRNA by proteins such as Dicer, and performs the same function as siRNA, and the siRNA sequence that performs the function can be the same as the aforementioned siRNA sequence.

[0065] In a preferred embodiment, the functional sequence used for knockdown in the shRNA is preferably ATACTCCATGATTAGCAATGG (SEQ ID NO. 3), TGGAGGTAGAGTACCGTGGTT (SEQ ID NO. 4).

[0066] In a preferred embodiment, miRNA refers to 21 to 23 non-coding RNAs that regulate gene expression after transcription by promoting the degradation of target RNA or by inhibiting its translation.

[0067] Modulators that reduce C8orf76 expression also include C8orf76 antibodies.

[0068] The above regulators can all inhibit the expression of C8orf76 protein.

[0069] In the present invention, the product also includes an anti-renal cancer drug, which contains a pharmaceutically acceptable carrier and a C8orf76 protein inhibitor, and the C8orf76 protein inhibitor can inhibit the expression of C8orf76.

[0070] The C8orf76 protein inhibitor is any one or more of a protease that degrades C8orf76 products, a nuclease that degrades C8orf76 products, or a regulator that reduces C8orf76 products. The C8orf76 protein inhibitor is an inhibitor that reduces the expression of C8orf76 in renal cancer cells compared to renal cancer cells not treated with the C8orf76 protein inhibitor.

[0071] The C8orf76 product reducing regulator is a C8orf76 antibody, and the C8orf76 antibody can directly reduce the C8orf76 protein level.

[0072] In a preferred embodiment, the carrier is any one or more of lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, ethylene glycol, water, saline or glucose aqueous solution.

[0073] In a preferred embodiment, the anti-renal cancer drug further comprises an antioxidant, and the antioxidant is any one of ascorbic acid, sodium sulfite or sodium bisulfate.

[0074] In a preferred embodiment, the anti-renal cancer drug further comprises an excipient, and the excipient is any one of corn starch, wheat starch, methyl cellulose, sodium carboxymethyl cellulose or gelatin.

[0075] The administration routes of the above anti-renal cancer drugs include oral, intravenous, parenteral, intramuscular, subcutaneous, intraperitoneal, intranasal, rectal or local administration.

[0076] In the present invention, the dosage of the drug of the present invention can be determined by factors such as the type of disease to be treated, the severity of the disease, the route of administration, the patient's age, gender, health status, etc. In a preferred embodiment, the dosage of the anti-renal cancer drug of the present invention can be 0.01 μg to 1000 mg / day for each patient.

[0077] The functions of C8orf76 and its expressed protein provided by the present invention are verified in detail below in combination with test examples.

[0078] Verification Example 1: C8orf76 is highly expressed in renal cancer tissue

[0079] Bioinformatics was used to retrieve the authoritative database TCGA-KIRC renal cancer dataset and compare the expression difference of C8orf76 between unpaired and paired cancer tissues and adjacent normal tissues. The results showed that the expression of C8orf76 in cancer tissues was higher than that in normal tissues (see Figure 1 , 2 ).

[0080] Using the database C8orf76 expression value and clinical diagnosis data, ROC was performed with specificity and sensitivity, and the relevant AUC was calculated, which confirmed that the expression difference of C8orf76 has important clinical value for the diagnosis of ccRCC (AUC = 0.820, see Figure 3 ).

[0081] The C8orf76 expression values, follow-up time, and clinical prognosis data of ccRCC patients in the existing public database were collected and sorted, and the patients were divided into a C8orf76 high expression group and a low expression group. At the same time, Log-rank P < 0.01 was used as a significant difference to draw a survival curve (see Figures 4 to 6 ).

[0082] The results showed that ccRCC patients with high expression of C8orf76 had a poor clinical prognosis, and their survival time was shorter than that of the low expression group.

[0083] Verification Example 2: C8orf76 protein is highly expressed in renal cancer tissue

[0084] The ccRCC cancer and adjacent tissues (10 pairs in total) collected in our center were selected, and the corresponding tissues were fully ground using a tissue grinder, and the tissues were fully lysed using RIPA strong lysis buffer (with protease inhibitors and phosphatase inhibitors added at a ratio of 1:100), and then allowed to stand on ice for 1 hour. After high-speed centrifugation at 4°C, the supernatant was collected, which was the tissue protein.

[0085] Use 10% SDS-PAGE gel, mix the prepared protein sample with 5× loading buffer in proportion, and place in a metal bath at 100℃ for 10min. Pre-stain protein marker, 5μL / lane, take a suitable volume of protein sample and add it to the lane. Use constant voltage 80V for electrophoresis for 30min, and constant voltage 120V until bromophenol blue reaches the bottom of the gel to terminate electrophoresis. Take out the gel, mark and transfer the membrane, immerse the gel in transfer buffer for 10min, cut the PVDF membrane and filter paper according to the size of the gel, place them in transfer buffer for 5min, and soak the PVDF membrane in methanol for 30 seconds before use.

[0086] Place the gel on the negative electrode side, assemble the transfer "sandwich" in order, add one layer at a time, and gently remove the bubbles with a glass rod. Place the transfer tank in ice, put the "sandwich" in accordance with the electrode, add the transfer buffer, 300mA constant current, and set the transfer time according to the molecular weight of the target protein. After the transfer is completed, place the transferred PVDF membrane in TBST buffer for use. Wash the membrane with TBST buffer for 5 minutes and shake slowly. Place the membrane in TBST containing 5% skim milk and block at room temperature for 1 to 2 hours. Wash the membrane 3 times with TBST, 5 minutes / time, place the membrane in a hybridization bag, add an appropriate amount of primary antibody diluted with antibody diluent, and incubate at 4°C with slow shaking overnight. Wash the membrane 3 times with TBST, 5 minutes / time. Place the membrane in a hybridization bag, add an appropriate amount of secondary antibody diluted with TBST, and incubate at 37°C for 1 hour.

[0087] Wash the membrane 3 times with TBST, 15 min / time. Prepare the developer according to the instructions of the developer kit, enter the darkroom, place the membrane in the hybridization bag, evenly apply an appropriate amount of developer, close the bag, remove the bubbles in the bag, fix the hybridization bag in the dark box, put the film close to the hybridization bag, do not leave any bubbles, close the dark box, use the appropriate exposure time, scan the film and analyze the image. The antibody dilution ratio refers to the antibody instructions.

[0088]

[0089] Table 1

[0090] Table 1 shows the corresponding antibodies for immunoblotting. Figure 7 , Figure 7 This is the immunoblot electrophoresis of C8orf76 after tissue protein extraction from renal cancer and adjacent tissue specimens obtained in our research center.

[0091] The results showed that the content of C8orf76 in cancer tissue (T) was significantly higher than that in adjacent paracancerous tissue (N).

[0092] Verification Example 3: C8orf76 is highly expressed in renal cancer cells

[0093] Normal renal tubular epithelial cells HK-2 and renal cancer cell lines (786-O, 769-P, Caki-1, A498) were cultured and sufficient amount of cells were collected for total RNA extraction.

[0094] 1) RNA extraction and cDNA preparation:

[0095] RNA was extracted using the Trizol method, and the RNA concentration was determined using NANODROP2000.

[0096] Preparation of cDNA:

[0097] a Reaction to remove genomic DNA:

[0098] Prepare PCR reaction solution on ice according to Table 1 and set the PCR instrument to 42°C for 2 min.

[0099] Table 2 shows the PCR reaction system for removing genomic DNA:

[0100]

[0101] Table 2

[0102] bReverse transcription reaction:

[0103] After the first step of the reaction, add the components listed in Table 2 to the PCR reaction tube in sequence, and set the PCR instrument to 37°C, 15 min; 85°C, 7 s; and store at 4°C. Dilute the reverse transcription reaction product 20 times and place it in a -20°C refrigerator.

[0104] Table 3 is the reverse transcription PCR reaction system:

[0105]

[0106] Table 3

[0107] 2) RT-qPCR detection

[0108] Quantitative primers were designed through the primer bank online design website, and primer specificity was analyzed using primer blast on the NCBI website. The primers were synthesized in the company (sequence information is shown in Table 3). The extracted RNA was reverse transcribed into cDNA, and the reverse transcribed cDNA was diluted 10 times as a template. Three replicates were set for each group of samples. The SYBR PremixEx TaqTM kit was used, and the amplification program was 95℃ pre-deformation for 2min; 94℃ denaturation for 30s, 60℃ annealing for 10s, and 72℃ extension for 10s, for a total of 40 cycles. The △△CT method was used to analyze the quantitative qPCR results.

[0109] Table 4 shows the RT-qPCR primer sequences:

[0110]

[0111] Table 4

[0112] Results Figure 8 , Figure 8 This is a qPCR result diagram of the difference in C8orf76 expression at the mRNA level between the normal renal tubular epithelial cell line (HK-2) and the ccRCC cell line. The figure shows that the C8orf76 mRNA content in 786-O, 769-P, and A498 renal cancer cell lines is significantly higher than that in the normal renal tubular epithelial cell line HK-2.

[0113] Verification Example 4: C8orf76 protein is highly expressed in renal cancer cells

[0114] The protein content of C8orf76 in the cell line was detected. The protein preparation method and immunoblotting technique were the same as those described in Validation Example 2. The results are shown in Fig. 9 shown.

[0115] Fig. 9 It was shown that the C8orf76 protein content in 786-O, 769-P, and A498 renal cancer cell lines was significantly higher than that in the normal renal tubular epithelial cell line HK-2.

[0116] The technical solution provided by the present invention is described in detail below in conjunction with embodiments.

[0117] Example 1: C8orf76 gene knockdown inhibits metastasis and proliferation of renal cancer cells

[0118] 1. Establishment of C8orf76 gene lentiviral knockdown cell line

[0119] Day 1:

[0120] Prepare cells: Culture cells to the logarithmic growth phase, digest with trypsin, count cells in the control group and knockdown group, resuspend in cell culture medium and culture equal numbers of cells in 10 cm cell culture dishes. This inoculum of 786-O cells can grow to 80% to 90% confluence on the third day after infection.

[0121] the next day:

[0122] (1) Prepare lentiviral particles - constructed C8orf76 gene RNA interference lentiviral vector particles (LV-C8orf76-RNAi) and control group lentiviral vector particles, calculate the amount of lentiviral particles required, take out the lentiviral particles frozen at -80°C, and thaw them in an ice bath. The functional sequences C8orf76-RNAi used to knock down C8orf76 are ATACTCCATGATTAGCAATGG (SEQ ID NO.3) and TGGAGGTAGAGTACCGTGGTT (SEQ ID NO.4), and the insertion sequence of the control group is TAAGACTACGGCGCGAAGC (SEQ: NC).

[0123] (2) Infect target cells: Take out the cells from the incubator and observe the cell growth status and cell confluence under a microscope; if the cells are in good condition, start the experiment:

[0124] A. Use a pipette to carefully remove the old culture medium from the 10 cm cell culture dish and add new complete culture medium;

[0125] B. Add the calculated lentiviral particles to the cells, place the culture plate flat on the workbench, and gently mix in a figure-8 pattern;

[0126] C. After mixing, place the cell culture plate at 37°C and 5% CO 2 Incubator, overnight culture.

[0127] Day 3:

[0128] Replace the culture medium: 12 to 16 hours after infection, aspirate the culture medium containing lentiviral particles, add 1640 complete culture medium containing 10% serum to the culture plate again, and continue culturing.

[0129] Day 4:

[0130] Continue to culture the cells and observe whether the cell status is abnormal.

[0131] Day 5:

[0132] Observe (evaluate) the infection efficiency of lentiviral particles: cover the 10 cm cell culture dish tightly, clean the outer wall of the culture plate with 75% ethanol, observe the fluorescence under an inverted fluorescence microscope, take pictures and estimate the infection efficiency of lentiviral particles on cells.

[0133] Day 6: When the number of fluorescent cells was found to be more than 50%, puromycin was used to screen positive cells, and C8orf76 gene lentiviral knockout 786-O cells, namely shC8orf76A, B, were obtained.

[0134] The fluorescence was observed by inverted fluorescence microscope. If the number of fluorescent cells was above 50%, the C8orf76 gene lentivirus successfully infected 786-O cells. The method for establishing 769-P stable knockdown C8orf76 cell line was the same.

[0135] 2. Determination of the interference efficiency of lentiviral knockdown of C8orf76 gene in 786-O cells

[0136] The shNC and shC8orf76-A, B cell lines were collected for total RNA extraction and C8orf76 mRNA content detection, and the method was the same as described in Verification Example 3. The shNC and shC8orf76-A, B cell lines were collected for total protein extraction and C8orf76 protein content detection, and the method was the same as described in Verification Example 2. The 769-P cell line method was the same.

[0137] Depend on Fig.10 , 11 It can be seen that compared with shNC, the expression levels of C8orf76 mRNA and protein of shC8orf76 were significantly decreased, that is, the C8orf76 gene lentiviral knockdown 786-O and 769-P cell lines were successfully established in this example.

[0138] 3. C8orf76 knockdown inhibits ccRCC cell proliferation

[0139] Single cell suspensions of the following cells were prepared respectively: C8orf76 gene lentiviral knockdown 786-O cells (shC8orf76-A, B) and their control group 786-O cells (shNC). Each group was seeded at a density of 5000 cells / well in a 96-well plate, and three replicates were set for each group. 10 μL CCK8 reagent was added to each well at different time periods, and incubated at 37°C in the dark for 1 h. The absorbance value was detected at a wavelength of 450 nm using an ELISA reader, and growth curves were prepared using GraphPad Prism 9.0 to detect cell proliferation at 0 h, 24 h, 48 h, 72 h, and 96 h in the shNC and shC8orf76-A, B groups, respectively (see Fig.12 ). The shNC and shC8orf76-A, B in 769-P cell line were operated as above, and the results are shown in Fig.13 .

[0140] Depend on Fig.12 and Fig.13 The results showed that knockdown of C8orf76 significantly inhibited the proliferation ability of renal clear cell carcinoma cells.

[0141] 4. C8orf76 knockdown inhibits ccRCC cell migration

[0142] Transwell migration assay: Stable cell lines were screened for 786-O cells (shC8orf76) knocked down by lentivirus of C8orf76 and their control group 786- cells (shNC), digested and collected, washed three times with PBS, resuspended in serum-free medium, counted under a microscope, and a single-cell suspension with a final concentration of 7×104 cells / mL was prepared. 100 μL of this single-cell suspension was added to the upper chamber of the chamber, and 600 μL of PRMI-1640 medium containing 10% FBS was added to the lower chamber of the chamber.

[0143] Each group has 3 parallel chambers. Cell culture: 37°C, 5% CO 2 The cells were cultured under the same conditions for 24 hours. The chamber was removed, and the cells on the upper side of the chamber membrane were wiped off with a cotton swab. The cells were fixed with 4% paraformaldehyde for 30 minutes and then stained with 5% crystal violet solution. More than three fields of view were selected and the number of cells penetrating the membrane was counted under a 100x optical microscope, and statistical analysis was performed.

[0144] Depend on Fig.14 , 15 The results showed that knockdown of C8orf76 significantly inhibited the migration ability of ccRCC cells.

[0145] 5. In vivo testing

[0146] Establishment of nude mouse subcutaneous heterotopic tumor model

[0147] 1) Cell preparation: Screen and expand the 786-O cell line with stable knockdown of C8orf76, prepare 4×10 cells per nude mouse 6 Cells (including control group and knockdown group) were collected in sterile EP tubes and resuspended in PBS for use;

[0148] 2) Preparation of nude mice: 3-4 week old BALB / c-nu nude mice were selected as experimental subjects and raised in the SPF-grade breeding room of the animal platform of our research center, and all aspects of breeding were strictly ensured to be sterilized and aseptic;

[0149] 3) Model establishment: Disinfect the injection area (subcutaneous on the right upper limb) with 5% iodine solution, mix the cells, and extract about 150 mL of cell suspension (4×10 6 ) was injected subcutaneously, and the weight was marked, weighed and recorded after completion;

[0150] 4) Measurement and observation: nude mice were observed weekly, the length and width of the subcutaneous transplanted tumors were measured, and body weight was weighed and recorded;

[0151] 5) Specimen collection: After 4 weeks, the nude mice were killed in strict accordance with animal ethics requirements, and the subcutaneous heterotopic transplanted tumors were removed, and the size and weight were measured and photographed. Note: The formula for calculating the volume of the transplanted tumor is: Volume = 0.5 × L × W 2 (W stands for wide diameter, L stands for long diameter);

[0152] 6) Specimen storage: Place the ex vivo transplanted tumor into sterile cell cryopreservation tubes, mark them according to the experimental groups, prepare two copies for each group, and store them in a -80°C refrigerator and at room temperature with paraformaldehyde fixation for future use.

[0153] Depend on Fig.16 The results showed that compared with the control group, knockdown of C8orf76 significantly inhibited tumor growth, and the volume and weight of tumors in the knockdown group were significantly reduced.

[0154] Embodiment 2:

[0155] Based on the pocket sitel (key amino acids LEU135 / THR149 / ASN166 / GLN144) predicted by Schrödinger software, computer virtual screening was performed in the bioactive drug library (containing 23,000 compounds) and the drug-like library (containing 70,000 compounds). Based on the molecular docking score, the potential compound 1,2,3,6-Tetragalloylglucose that can bind to the C8orf76 protein and inhibit its function was obtained. The binding mode of this compound with C8orf76 is shown in the figure below. Figures 17 to 20 As shown ( Fig.17 is a cartoon picture, he 18 is a surface picture, Fig.19 For 2D images, Fig. 20 for 3D images).

[0156] When 1,2,3,6-Tetragalloylglucose inhibitor was added to 786-O cells after exogenous overexpression of C8orf76 protein (OE-C8orf76), the growth of cells was restricted to a certain extent, suggesting that 1,2,3,6-Tetragalloylglucose can reduce the proliferation ability of tumor cells 786-O by inhibiting the function of C8of76 protein. Fig.21 shown.

[0157] It can be seen from Example 1 that C8orf76 can be used as a therapeutic target in the preparation of drugs for treating renal cancer. After using shRNA to knock out or silence C8orf76, the proliferation and migration abilities of 786-O and 769-P cells were significantly inhibited after lentiviral knockdown of the C8orf76 gene, and tumor growth was significantly inhibited at the animal level.

[0158] It can be seen from Example 2 that C8orf76 can be used as a therapeutic target in the preparation of drugs for treating renal cancer. After using the C8orf76 protein inhibitor 1,2,3,6-Tetragalloylglucose, the 1,2,3,6-Tetragalloylglucose inhibitor can bind to the C8orf76 protein and inhibit its function, and the growth of renal cancer cells is restricted to a certain extent. By inhibiting the function of the C8of76 protein, the proliferation ability of tumor cells 786-O is weakened.

[0159] This invention proposes and verifies for the first time that C8orf76 protein is a new target for the diagnosis and gene therapy of ccRCC, which is of great significance for the screening of new anti-renal cancer drugs and also provides a new idea for the diagnosis and treatment of renal cancer.

[0160] The above embodiments are only exemplary embodiments of the present application and are not intended to limit the present application. The protection scope of the present application is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present application within the essence and protection scope of the present application, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present application.

Claims

1. Application of a therapeutic target in a product for inhibiting the metastasis and proliferation of renal cancer cells, characterized in that: The nucleotide sequence of the C8orf76 therapeutic target is shown in SEQ ID NO.1; The expression protein of the C8orf76 therapeutic target is C8orf76 protein; The amino acid sequence of the C8orf76 protein is shown in SEQ ID NO.

2.

2. The use of a therapeutic target in a product for inhibiting the metastasis and proliferation of renal cancer cells according to claim 1, characterized in that: The kit contains reagents for knocking out or silencing the C8orf76 therapeutic target; The reagent includes siRNA, shRNA or miRNA; The functional sequence of the shRNA is shown in SEQ ID NO.3 or SEQ ID NO.

4.

3. The use of a therapeutic target in a product for inhibiting the metastasis and proliferation of renal cancer cells according to claim 2, characterized in that: The product in question is an anti-kidney cancer drug; The anti-renal cancer drug comprises a pharmaceutically acceptable carrier and a C8orf76 protein inhibitor.

4. The use of a therapeutic target in a product for inhibiting the metastasis and proliferation of renal cancer cells according to claim 3, characterized in that: The C8orf76 protein inhibitor is any one or more of a protease that degrades C8orf76 products, a nuclease that degrades C8orf76 products, or a regulator that reduces C8orf76 products.

5. The use of a therapeutic target in a product for inhibiting the metastasis and proliferation of renal cancer cells according to claim 3, characterized in that: The C8orf76 protein inhibitor is a 1,2,3,6-Tetragalloylglucose inhibitor.

6. The use of a therapeutic target in a product for inhibiting the metastasis and proliferation of renal cancer cells according to claim 3, characterized in that: The C8orf76 product reducing regulator is a C8orf76 antibody; The carrier is any one or more of lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, ethylene glycol, water, saline or glucose aqueous solution; The anti-renal cancer drug also includes an antioxidant; Wherein, the antioxidant is any one of ascorbic acid, sodium sulfite or sodium bisulfate.

7. The use of a therapeutic target in a product for inhibiting the metastasis and proliferation of renal cancer cells according to claim 3, characterized in that: The anti-renal cancer drug also includes an excipient; Wherein, the excipient is any one of corn starch, wheat starch, methyl cellulose, sodium carboxymethyl cellulose or gelatin.

8. The use of a therapeutic target in a product for inhibiting the metastasis and proliferation of renal cancer cells according to claim 3, characterized in that: The administration routes of the anti-renal cancer drug include oral, intravenous, parenteral, intramuscular, subcutaneous, intraperitoneal, intranasal, rectal or local administration.

9. The use of a therapeutic target in a product for inhibiting the metastasis and proliferation of renal cancer cells according to claim 3, characterized in that: The dosage of the C8orf76 protein inhibitor is 0.01 μg to 1000 mg / day for each patient.

10. The use of a therapeutic target in a product for inhibiting the metastasis and proliferation of renal cancer cells according to claim 1, characterized in that: The renal cancer cell is ccRCC.

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