Application of LINC02539 in the diagnosis and treatment of renal cancer

Abstract

The present invention discloses a biomarker for the diagnosis and treatment of renal cancer, specifically LINC02539. The present invention first discovered that the differential expression of LINC02539 is related to the occurrence and development of renal cancer. In patients with renal cancer, the expression of LINC02539 is upregulated, suggesting that intervening in the expression of LINC02539 gene may become a new approach to the treatment of renal cancer. For further verification, it was found that the proliferation of renal cancer cells and the number of cell-formed clone colonies can be significantly reduced by reducing the expression of LINC02539 through inhibitors.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of LINC02539 in the diagnosis and treatment of renal cancer. Background Art

[0002] Renal cell carcinoma (RCC) is a common type of urinary system malignancy originating from renal tubular epithelial cells. Among new cancer cases in men and women, the proportion of renal cell carcinoma is 3% and 5%, respectively. Clear cell renal cell carcinoma (ccRCC) is the most common and most aggressive renal cancer, accounting for 80% of new RCC cases and has a high mortality rate. Other subtypes include papillary carcinoma, chromophobe carcinoma, and collecting duct carcinoma. Due to its high metastasis rate, difficult diagnosis, and poor prognosis are typical characteristics of renal cell carcinoma. Currently, surgical resection is the first-line treatment for localized renal cell carcinoma. However, even after radical nephrectomy, about 30% of patients will still experience local or distant recurrence. The treatment of renal malignancies is an ongoing challenge, so revealing the pathogenesis of renal cell carcinoma and exploring effective treatments are the current focus of renal cell carcinoma research.

[0003] LncRNA is a type of transcript that is longer than 200 nucleotides and cannot encode proteins or polypeptides. In recent years, with the widespread application of high-throughput sequencing technology, long non-coding RNAs that were originally considered to be "noise" of genomic transcription have been shown to play an important biological role in many diseases. A large number of studies have found that LncRNAs participate in tumor carcinogenesis and protein-coding gene expression disorders by binding to chromatin-modifying proteins and changing their states, while binding to transcription factors, RNA-binding proteins and miRNAs. It is increasingly valued in the biological behavior of tumors, providing a new way to diagnose and treat tumors. At present, a variety of LncRNAs have been found to be associated with renal cell carcinoma, such as LncRNA HIF1A-AS2, LncRNA 00312, LncRNA SNHG12, etc. At present, the research on the regulatory mechanism of LncRNA in renal cell carcinoma has not been elucidated. Exploring LncRNA plays an important role in the diagnosis, prevention and treatment of renal cell carcinoma, and can provide a new direction for gene-targeted therapy of renal cell carcinoma. Summary of the invention

[0004] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a biomarker related to the occurrence and development of renal cancer, and to achieve early diagnosis of renal cancer and precise molecular targeted treatment of renal cancer by detecting the biomarker.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] The present invention provides use of a reagent for detecting LINC02539 in preparing a product for diagnosing renal cancer.

[0007] The reagents include reagents for detecting the expression level of LINC02539 using RT-PCR, real-time quantitative PCR, in situ hybridization, northern blotting, chips or high-throughput sequencing platforms.

[0008] Furthermore, the reagents for detecting LINC02539 by real-time quantitative PCR include at least a pair of primers for specifically amplifying LINC02539.

[0009] Furthermore, the reagent comprises:

[0010] A probe that specifically recognizes LINC02539; or

[0011] Primers that specifically amplify LINC02539.

[0012] Furthermore, the primer sequences for specifically amplifying LINC02539 are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0013] The present invention provides a product for diagnosing renal cancer, comprising a reagent for detecting the expression level of LINC02539 in a sample.

[0014] The product includes a chip, a preparation or a kit. The chip includes a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, and the oligonucleotide probe includes an oligonucleotide probe for LINC02539 for detecting the transcription level of LINC02539; the kit includes a primer or a chip for detecting the transcription level of LINC02539.

[0015] The solid phase carrier can be made of various commonly used materials in the field of gene chips, such as but not limited to nylon membranes, glass slides or silicon slices modified with active groups (such as aldehyde groups, amino groups, etc.), unmodified glass slides, plastic sheets, etc.

[0016] The gene detection kit or gene chip of the present invention can be used to detect the expression levels of multiple genes (for example, multiple genes related to renal cancer) including the LINC02539 gene. The simultaneous detection of multiple markers of renal cancer can greatly improve the accuracy of renal cancer diagnosis.

[0017] Further, the reagent for detecting the level of LINC02539 in the sample includes:

[0018] A probe that specifically recognizes LINC02539; or

[0019] Primers that specifically amplify LINC02539.

[0020] Furthermore, the primer sequences for specific amplification of LINC02539 are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0021] The present invention provides use of LINC02539 in preparing a pharmaceutical composition for treating renal cancer.

[0022] Furthermore, the pharmaceutical composition includes an inhibitor of LINC02539, and / or other drugs compatible with the inhibitor and a pharmaceutically acceptable carrier and / or excipient, wherein the inhibitor can inhibit the expression of LINC02539 or a substance involved in an upstream or downstream pathway of LINC02539. The inhibitor can act at the DNA level or the RNA (i.e., gene product) level.

[0023] Furthermore, the inhibitor is siRNA targeting LINC02539.

[0024] The present invention screens effective siRNA sequences by performing a large number of comparison analyses to find the best effective fragments. In a specific embodiment of the present invention, the inventors designed and synthesized a variety of siRNA sequences, and verified them by transfecting renal cancer cell lines with transfection reagents. As a result, interfering molecules with better interference effects were detected, which have the sequences shown in SEQ ID NO.9 and SEQ ID NO.10, respectively. Further experiments at the cell level proved that the inhibition efficiency was very high for cell experiments.

[0025] The nucleic acid inhibitors of the present invention, such as siRNA, can be chemically synthesized or prepared by transcribing an expression cassette in a recombinant nucleic acid structure into a single-stranded RNA. Nucleic acid inhibitors such as siRNA can be delivered into cells by using appropriate transfection reagents or by using a variety of techniques known in the art.

[0026] Those skilled in the art will recognize that the utility of the present invention is not limited to quantifying the gene expression of any specific variant of the marker gene of the present invention. In a specific embodiment, as a non-limiting example, the marker gene LINC02539 has the sequence shown in the current international public nucleic acid sequence database GeneBank LINC02539 gene (NC_000001.11).

[0027] In the present invention, the term "probe" refers to a molecule that can bind to a specific sequence or subsequence or other part of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that can bind to another polynucleotide (often referred to as a "target polynucleotide") through complementary base pairing. Depending on the stringency of the hybridization conditions, the probe can bind to a target polynucleotide that lacks complete sequence complementarity with the probe. The probe can be directly or indirectly labeled, including primers. Hybridization methods include, but are not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

[0028] In the present invention, pharmaceutically acceptable carriers include (but are not limited to): diluents, excipients such as lactose, sodium chloride, glucose, urea, starch, water, etc., fillers such as starch, sucrose, etc.; binders such as monosyrup, glucose solution, starch solution, cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; wetting agents such as glycerol; disintegrants such as dry starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds, sodium lauryl sulfate, etc.; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, hexadecanol, etc.; wetting agents such as glycerol, starch, etc.; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin and soap clay, etc.; lubricants such as talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, etc.

[0029] In the present invention, the pharmaceutical composition can be prepared using various additives, such as buffers, stabilizers, bacteriostatic agents, isotonic agents, chelating agents, pH control agents and surfactants.

[0030] The pharmaceutical composition of the present invention can be administered orally, parenterally, by inhalation spray, topical administration, rectal administration, nasal administration, buccal administration, vaginal administration or by implanted drug storage device administration. Oral administration or injection is preferred. The pharmaceutical composition of the present invention may contain any commonly used non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle. In some cases, medicinal acid, alkali or buffer can be used to adjust the pH of the preparation to improve the stability of the compound or its dosage form. The term parenteral used in the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intracorporeal, intralesional, intralesional, and intracranial injection or infusion techniques. As long as the target tissue can be reached, the pharmaceutical composition of the present invention can be administered to the receptor by any route.

[0031] The pharmaceutical composition of the present invention can be orally administered in the form of any oral dosage form, including but not limited to capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. For oral tablets, common carriers include lactose and corn starch. Lubricants such as magnesium stearate are generally also added. For oral administration in capsule form, suitable diluents include lactose and anhydrous corn starch. When aqueous suspensions and / or emulsions are administered orally, the active ingredient can be suspended or dissolved in an oil phase and combined with an emulsifier and / or suspending agent. If necessary, some sweeteners and / or flavoring agents and / or coloring agents can be added. Where appropriate, the dosage unit formulation for oral administration can be microencapsulated. For example, by coating or embedding the particulate matter in a polymer, wax, etc., the preparation can also be prepared to extend or maintain release. The pharmaceutical composition of the present invention can be used to reduce endogenous LINC02539 overexpression, thereby treating renal cancer caused by upregulation of LINC02539 expression by reducing the expression of LINC02539.

[0032] In the present invention, the compound that inhibits the expression of LINC02539 can be administered to a subject as naked RNA together with a delivery agent as a nucleic acid (such as a recombinant plasmid or a viral vector), wherein the nucleic acid comprises a sequence that inhibits the expression of LINC02539. The delivery agent can be a lipophilic agent, a polycation, a liposome, and the like.

[0033] The pharmaceutical composition of the present invention may further comprise one or more anticancer agents. In a specific embodiment, the pharmaceutical composition comprises at least one compound that inhibits LINC02539 gene expression and at least one chemotherapeutic agent. Chemotherapeutic agents used in the present invention include, but are not limited to, microtubule activators, alkylating agents, anti-neoplastic antimetabolites, platinum compounds, DNA-alkylating agents, anti-tumor antibiotics, antimetabolites, microtubule stabilizers, microtubule destabilizers, hormone antagonists, topoisomerase inhibitors, protein kinase inhibitors, HMG-COA inhibitors, CDK inhibitors, cell cycle protein inhibitors, caspase inhibitors, metalloproteinase inhibitors, antisense nucleic acids, triple-stranded helix DNA, nucleic acid aptamers, and molecularly modified viruses, bacteria and exotoxin agents.

[0034] The drug of the present invention can also be used in combination with other drugs for treating renal cancer. Other therapeutic compounds can be administered simultaneously with the main active ingredient, or even in the same composition. Other therapeutic compounds can also be administered separately in a separate composition or in a dosage form different from the main active ingredient. Partial doses of the main ingredient can be administered simultaneously with other therapeutic compounds, while other doses can be administered separately. During the treatment process, the dosage of the pharmaceutical composition of the present invention can be adjusted according to the severity of the symptoms, the frequency of recurrence, and the physiological response of the treatment plan.

[0035] The pharmaceutical composition of the present invention can be a pharmaceutical composition for topical administration and can be formulated into ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.

[0036] The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" of the present invention refers to an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dosage level can be determined based on factors including the severity of the disease, the activity of the drug, the age, weight, health, sex of the patient, the sensitivity of the patient to the drug, the administration time, administration route and excretion rate of the composition of the present invention used, the treatment time, the elements of the drug used in combination or simultaneously with the composition of the present invention used, and other factors known in the medical field. The pharmaceutical composition of the present invention can be administered as a single therapeutic agent, or administered in combination with other therapeutic agents, and can be administered sequentially or simultaneously with previous therapeutic agents. In addition, it can be administered once or multiple times. It is important that all of the above factors are taken into consideration, and the drug is administered in the amount that can achieve the maximum effect with the minimum amount without side effects.

[0037] The term "treatment" means not for the purpose of curing, but to slow down (reduce) the targeted pathological condition or disorder or prevent recurrence. If a patient is successfully "treated" after receiving a therapeutically effective amount of a therapeutic agent, the patient shows an observable and / or measurable reduction or disappearance of one or more signs and symptoms of a specific disease. For example, a significant reduction in the number of cancer cells or the disappearance of cancer cells, a reduction in tumor size; inhibition (i.e., slowing down to some extent, and preferably stopping) of tumor metastasis; inhibition of tumor growth to some extent; an increase in the time of reducing and / or alleviating to some extent one or more symptoms associated with a specific cancer; reduced morbidity and mortality, and improved quality of life. The reduction of signs or symptoms of the disease can be perceived by the patient. Treatment can achieve a complete response - defined as the disappearance of all signs of cancer, or a partial response - a reduction in tumor size, preferably a reduction of more than 50%, more preferably 75%. If the patient feels that the disease is stable, the patient is also considered to be treated.

[0038] In the present invention, the term "sample" includes (but is not limited to) cells, tissues, organs, body fluids (blood, lymph, etc.), digestive fluid, sputum, alveolar bronchial washings, urine, feces, etc. Preferably, the sample is tissue or blood.

[0039] The present invention also provides the use of LINC02539 in screening substances to be screened for preventing or treating renal cancer.

[0040] Furthermore, the step of screening the substance to be screened for preventing or treating renal cancer comprises:

[0041] Treating a system expressing or containing the LINC02539 gene with a substance to be screened; and

[0042] Detecting the expression of the LINC02539 gene in the system;

[0043] Among them, if the substance to be screened can reduce the expression or activity of the LINC02539 gene (preferably significantly reduced, such as 20% or more lower, preferably 50% or more lower, and more preferably 80% or more lower), it indicates that the substance to be screened is a substance to be screened for preventing or treating renal cancer.

[0044] Furthermore, the above-mentioned systems include (but are not limited to): cell systems, subcellular systems, solution systems, tissue systems, organ systems or animal systems.

[0045] Compared with the prior art, the present invention provides a product for diagnosing early renal cancer, so that patients can be treated at an early stage, thereby improving survival rate and life treatment. The present invention also provides a treatment method and a pharmaceutical composition to achieve precise molecular treatment of renal cancer. The present invention provides a method for screening substances to be screened for preventing or treating renal cancer, which plays an important role in the diagnosis, prognosis and prediction of renal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The figure shows the expression of LINC02539 in renal cancer patients detected by QPCR;

[0047] Figure 2 The figure shows the expression of LINC02539 in renal cancer cells detected by QPCR;

[0048] Figure 3 This is a diagram of the effect of transfected siRNA on the expression of LINC02539 in ACHN renal cancer cells detected by QPCR;

[0049] Figure 4 This is a diagram of using CCK8 to detect the effect of LINC02539 on the proliferation of ACHN renal cancer cells. DETAILED DESCRIPTION

[0050] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the examples where specific conditions are not specified are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under the conditions recommended by the manufacturer.

[0051] Example 1: Screening for gene markers associated with renal cancer

[0052] 1. Sample collection

[0053] Cancer tissues and adjacent tissues were collected from 50 patients with renal cancer, and all patients gave informed consent. The acquisition of all the above specimens was approved by the organization's ethics committee.

[0054] 2. Preparation of RNA Samples

[0055] Tissue RNA was extracted using QIAGEN's tissue RNA extraction kit according to the specific steps in the instruction manual.

[0056] 3. Quality analysis of RNA samples

[0057] The concentration and purity of the extracted RNA were tested by Nanodrop 2000, the integrity of RNA was tested by agarose gel electrophoresis, and the RIN value was determined by Agilent 2100. The concentration was ≥ 200 ng / μl, and the OD260 / 280 was between 1.8 and 2.2.

[0058] 4. Removal of rRNA

[0059] Ribosomal RNA was removed from total RNA using the Ribo-Zero kit.

[0060] 5. Construction of cDNA library

[0061] The cDNA library was constructed using Illumina's Truseq RNA sample Prep Kit, and the specific operations were performed according to the instructions.

[0062] 6. Sequencing

[0063] The cDNA library was sequenced using the HiSeq4000 sequencing platform, and the specific operations were performed according to the instructions.

[0064] 7. High-throughput transcriptome sequencing data analysis

[0065] The R package DESeq2 was used to perform bioinformatics analysis on the original count data, and lncRNAs that were not easily detected were deleted. The screening criteria for differentially expressed lncRNAs were: FDR < 0.05, abs (log2FC) > 2.

[0066] 7. Results

[0067] RNA-seq results showed that the expression level of LINC02539 in renal cancer tissues was significantly higher than that in adjacent adjacent tissues.

[0068] Example 2: QPCR sequencing to verify the differential expression of LINC02539 gene

[0069] 1. Large-sample QPCR verification of differential expression of LINC02539 gene. According to the sample collection method in Example 1, 50 samples of renal cancer tissue and 50 samples of adjacent tissue were collected.

[0070] 2. The RNA extraction procedure is the same as in Example 1.

[0071] 3. Reverse transcription:

[0072] A 20 μl reaction system was used, 1 μg of total RNA was taken from each sample as template RNA, and the following components were added to the PCR tube: DEPC water, premixed reagent, template RNA. 37°C for 15 min, 50°C for 5 min, 98°C for 5 min, and maintained at 4°C.

[0073] (3) QPCR amplification test

[0074] Primers were designed based on the sequences of LINC02539 gene and housekeeping gene GAPDH, and the primer sequences were synthesized by Shanghai Bioengineering. The designed primer sequences are as follows:

[0075] The primer sequences for the LINC02539 gene are:

[0076] Forward primer: 5'-CTCATTTGGGCGAAATCCTATCA-3' (SEQ ID NO.1)

[0077] Reverse primer: 5'-TTCTTCCATTGCCTGGTGCG-3' (SEQ ID NO. 2)

[0078] The primer sequences for the housekeeping gene GAPDH are:

[0079] Forward primer: 5'-GAAAGCCTGCCGGTGACTAA-3' (SEQ ID NO.3)

[0080] Reverse primer: 5'-GCCCAATACGACCAAATCAGAG-3' (SEQ ID NO. 4)

[0081] Prepare 10 μl of the following reaction system: 5 μl of SYBR Green polymerase chain reaction system, 2 μl of each forward and reverse primer (5 μM), and 1 μl of template cDNA. All operations were performed on ice. Three parallel tubes were set up for each sample, and all amplification reactions were repeated more than three times to ensure the reliability of the results. The amplification program was: 95°C 60s, (95°C 10s, 60°C 30s) × 40 cycles.

[0082] SYBR Green was used as a fluorescent marker and PCR reaction was performed on a 7500 Fast fluorescent real-time quantitative PCR instrument. The target bands were determined by melting curve analysis and electrophoresis, and relative quantification was performed by the ΔΔCT method.

[0083] 3. Statistical methods

[0084] The experiments were repeated three times and the results were expressed as mean ± standard deviation. SPSS18.0 statistical software was used for statistical analysis. The difference between the two was analyzed using t-test, and it was considered statistically significant when P < 0.05.

[0085] 4. Results

[0086] The results are as follows Figure 1 As shown in the figure, compared with adjacent adjacent tissues, the expression of LINC02539 gene was upregulated in renal cancer tissues, and the difference was statistically significant (P<0.05), which was consistent with the results of RNA-sep.

[0087] Example 3: Differential expression of LINC02539 gene in renal cancer cell lines

[0088] 1. Cell culture

[0089] Human renal cancer cell lines ACHN, 786-0 and normal kidney cell line HK-2 were cultured in DMEM or 1640 containing 10% fetal bovine serum and 1% P / S in an incubator at 37°C, 5% CO2, and 90% relative humidity. The medium was changed every 2-3 days, and the cells were routinely digested and passaged using 0.25% trypsin containing EDTA.

[0090] 2. RNA Extraction

[0091] The RNA in the cells was extracted using Qiagen's cell RNA extraction kit, and the experimental operation was carried out according to the instruction manual.

[0092] 3. The specific steps of reverse transcription are the same as those in Example 2

[0093] 4. Statistical methods

[0094] The experiments were repeated three times and the results were expressed as mean ± standard deviation. SPSS18.0 statistical software was used for statistical analysis. The difference between the two was analyzed using t-test, and it was considered statistically significant when P < 0.05.

[0095] 5. Results

[0096] The results are as follows Figure 2As shown, compared with normal renal cell lines, the expression of LINC02539 gene was upregulated in renal cancer cells ACHN and 786-0, and the difference was statistically significant (P<0.05), which was consistent with the RNA-sep results.

[0097] Example 4: Silencing of LINC02539 gene

[0098] 1. Cell culture

[0099] The human renal cancer cell line ACHN was cultured, and the specific operation was the same as that in Example 3.

[0100] 2. siRNA Design

[0101] Interfering RNA was designed according to the sequence of LINC02539, and the experiment was divided into four groups: blank control group (ACHN), negative control group (siRNA-NC) and experimental group (siRNA1, siRNA2, siRNA3). The siRNA in the negative control group had no homology with the sequence of the LINC02539 gene. The designed siRNA sequences are as follows:

[0102] Negative control siRNA sequence (siRNA-NC):

[0103] The positive strand is 5'-ACCAAAUCAGAGAAUAAUCUA-3' (SEQ ID NO. 5),

[0104] The antisense strand is 5′-GAUUAUUCUCUGAUUUGGUCG-3′ (SEQ ID NO. 6);

[0105] siRNA1:

[0106] The positive strand is 5'-AAGAAUCUAGGUAACAGACAA-3' (SEQ ID NO. 7),

[0107] The antisense strand is 5′-GUCUGUUACCUAGAUUCUUAU-3′ (SEQ ID NO. 8);

[0108] siRNA2:

[0109] The positive strand is 5'-AAUUUAAAUUCUCUAACUCAC-3' (SEQ ID NO. 9),

[0110] The antisense strand is 5′-GAGUUAGAGAAUUUAAAUUAC-3′ (SEQ ID NO. 10);

[0111] siRNA3:

[0112] The positive strand is 5'-UAUUUUAUCCUUACAAAAGUC-3' (SEQ ID NO.11),

[0113] The antisense strand was 5'-CUUUUGUAAGGAUAAAAUAAA-3' (SEQ ID NO. 12). The cells were seeded into a six-well cell culture plate at 2×105 / well and cultured in a 37°C, 5% CO2 incubator for 24 hours. The cells were transfected according to the instructions of Lipofectamine 2000 (purchased from Invitrogen) in a DMEM medium without dual antibodies and containing 10% FBS.

[0114] 3. QPCR detection of transcription level of LINC02539 gene

[0115] 3.1 The specific steps for extracting total cell RNA are the same as those in Example 3.

[0116] 3.2 The reverse transcription step is the same as in Example 2.

[0117] 3.3QPCR amplification steps are the same as in Example 2.

[0118] 4. Statistical methods

[0119] The experiments were repeated three times, and the results were expressed as mean ± standard deviation. SPSS18.0 statistical software was used for statistical analysis. The difference between the LINC02539 gene expression group and the control group was analyzed using the t-test, and it was considered statistically significant when P < 0.05.

[0120] 5. Results

[0121] The results are as follows Figure 3 The results showed that compared with the cells transfected with empty siRNA-NC, the level of LINC02539 mRNA in the experimental group decreased, among which the interference effect of siRNA2 group was the most significant, and the difference was statistically significant (P<0.05).

[0122] Example 5: CCK8 detection of cell proliferation experiment

[0123] 1. Cell culture and transfection steps are the same as in Example 4

[0124] 2. CCK8 detection of cell proliferation

[0125] 1) ACHN cells in the logarithmic growth phase were seeded in 96-well plates, with 2×10 3 cells; the experiment was divided into groups (control group, siRNA-NC group, siRNA2 group), and each group had 6 replicate wells;

[0126] 2) Add 10 μl / well CCK8 reagent at 0h, 24h, 48h, and 72h after transfection;

[0127] 3) After 2 hours, use an ELISA reader to detect the absorbance value of A450.

[0128] 3. Statistical methods

[0129] The experiments were repeated three times and SPSS13.0 statistical software was used for statistical analysis. The differences between the two were tested using t-test, and were considered statistically significant when P < 0.05.

[0130] 4. Results

[0131] Figure 4 The results shown in the figure show that the cell growth rate in the siRNA2 group was significantly lower than that in the control group, and the difference was statistically significant (P<0.05). The above results indicate that knockout of LINC02539 can inhibit the growth of renal cancer cells.

[0132] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of reagents for detecting LINC02539 in the preparation of products for diagnosing renal cancer.

2. The use according to claim 1, characterized in that: The reagents include: A probe that specifically recognizes LINC02539; or Primers that specifically amplify LINC02539.

3. The use according to claim 2, characterized in that: The primer sequences for specifically amplifying LINC02539 are shown in SEQ ID NO.1 and SEQ ID NO.

2.

4. Use of an inhibitor of LINC02539 in the preparation of a pharmaceutical composition for treating renal cancer, characterized in that: The inhibitor is siRNA2 targeting LINC02539, and the sequence is: The positive strand is 5'-AAUUUAAAUUCUCUAACUCAC-3', The antisense strand is 5'-GAGUUAGAGAAUUUAAAUUAC-3'.

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