Use of miRNA-23c as a gene targeting METTL3 in preparation of drugs for preventing or treating bladder cancer

Abstract

The application discloses application of miRNA-23c as a gene targeting METTL3 in preparation of a medicine for preventing or treating bladder cancer. The application discloses a miRNA for inhibiting growth of bladder cancer, the miRNA-23c is complementary to the 3'-UTR of the mRNA of the METTL3 gene, can inhibit translation of the mRNA of a target gene or directly degrade the mRNA of the target gene. Fluorescent luciferase reporter gene analysis in an EJ cell line verifies that the METTL3 is a target gene of the miRNA-23c, then overexpression of the miRNA-23c in the EJ cell line finds that the miRNA-23c significantly down-regulates the mRNA level of the METTL3. The application first reports that the METTL3 can be a target gene of the miRNA-23c. The application provides certain application value in the aspect of using the miRNA as a drug target point for bladder cancer treatment.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, and particularly relates to application of miRNA-23c as a gene targeting METTL3 in preparation of a drug for preventing or treating bladder cancer. BACKGROUND

[0002] MicroRNA (miRNA) plays an important regulatory function in the growth, development and occurrence of various diseases of organisms, and has become a hotspot in current life science research. The miRNA is a kind of non-coding single-stranded small molecule RNA with a length of 21-22 nt, which degrades the target mRNA or inhibits the translation thereof through complementary pairing with the target mRNA. Studies have shown that the miRNA plays an important role in the occurrence of tumors, and the miRNA is likely to become a new approach for cancer treatment and diagnosis. The miRNA and the non-coding sequence (3'-UTR or 5'-UTR) of the target mRNA have a one-to-many relationship. In cancer cases, the expression of miRNA groups is often found to be disordered, and the change in the expression of the miRNA is closely related to the occurrence of cancer. In vitro experiments, the expression of one or more miRNAs can promote or inhibit the malignancy of cancer cells, and the miRNA that can promote the malignancy of cancer cells can be considered as a kind of cancer gene, and vice versa.

[0003] Bladder cancer (BC) accounts for the first place of the incidence of urogenital system tumors in China, and its incidence rate in western countries is only next to that of prostate cancer. According to statistics, bladder cancer ranks the sixth in the global male new cases in 2020, about 440,000. Surgical resection and chemotherapy are two major treatment strategies for bladder cancer. However, most bladder cancer patients are diagnosed as middle and late stages once found. Even the patients who receive active treatment still face the difficulties of recurrence, poor prognosis and low survival rate. Therefore, it is particularly urgent to deeply explore the molecular regulation mechanism of the occurrence and development of bladder cancer in order to provide a new intervention target and theoretical basis for clinical diagnosis and treatment. N6-methyladenosine (m6A) is the most abundant RNA modification in eukaryotic cells, accounting for more than 80% of methylation modification, and has the functions of regulating the splicing, positioning, transport, stability and translation efficiency of RNA. METTL3 catalyzing process modifies most m6A sites and is considered to be the most common m6A pathway. The m6A modification mediated by METTL3 is also a key promoter of EMT and metastasis of malignant tumors. The miRNA can become a new marker related to early diagnosis and progression of bladder cancer, and is helpful for accurate diagnosis and individualized treatment of bladder cancer. SUMMARY

[0004] The application aims to provide a new medical use of miRNA-23c, and the miRNA-23c can inhibit the expression of the METTL3 gene in bladder cancer, thereby providing important data for the development of drugs for bladder cancer.

[0005] To achieve the above-mentioned object, the application adopts the following technical scheme:

[0006] The application provides an application of miRNA-23c as a gene targeting METTL3 in the preparation of a drug for preventing or treating bladder cancer; the nucleotide sequence of the miRNA-23c is 5'-AUCACAUUGCCAGUGAUUACCC-3'.

[0007] Preferably, the drug is used for inhibiting the expression of the METTL3 gene in bladder cancer; or the drug is used for down-regulating the expression level of the METTL3 gene in bladder cancer.

[0008] In another aspect, the application further provides a drug preparation for preventing or treating bladder cancer, which comprises (1) an effective amount of miRNA-23c as an active ingredient, and (2) a pharmaceutically acceptable carrier.

[0009] Preferably, the drug preparation is in the form of a capsule, a tablet, a powder, an oral liquid, a pill, a tincture, a syrup or an injection.

[0010] The research idea of the application is further described as follows:

[0011] According to the qRT-PCR detection of the gene expression of 30 clinical samples of bladder cancer, it is found that the expression of a gene related to epithelial-mesenchymal transition is related to the occurrence and development of bladder cancer. The METTL3 is up-regulated at the mRNA and protein levels in bladder cancer cells. The differentially expressed miRNAs are screened through the TCGA bladder cancer data, the differential miRNAs of METTL3 are predicted through the multiMiR R software package, and the miRNAs upstream of METTL3 are predicted through the Starbase database, the intersection of the three is found to be the miRNAs that may mediate the METTL3 to participate in the regulation of bladder cancer, and it is found that the miRNA-23c can target the METTL3. The luciferase activity detection experiment and the overexpression analysis of the miRNA-23c prove that the miRNA-23c can target and inhibit the expression of the METTL3 gene.

[0012] The total RNA of cells is extracted by the Trizol reagent of a biological company, reverse transcription is performed, and the expression of the METTL3 in the clinical cases of bladder cancer is detected. It is found that, compared with the paracancerous tissues of the cases, the METTL3 is significantly increased in the bladder cancer.

[0013] According to the bioinformatics prediction, the 3'UTR of METTL3 is inserted into the 3'UTR end of the reporter plasmid Luciferase, and some sites of the segment are mutated, and the mutated segment is also inserted into the 3'UTR end of the reporter plasmid Luciferase, and WT and MUT Luciferase Reporter Plasmids are constructed, and the plasmid is transfected into bladder cancer cells with NC mimic, miRNA-23c mimic, NC inhibitor, and miRNA-23c inhibitor. 48 hours after transfection, a specific luciferase substrate is added, luciferase reacts with the substrate to produce fluorescence, and the intensity of the fluorescence can be detected to determine the activity of luciferase, and then it is judged whether miRNA-23c can interact with METTL3. It is shown that miRNA-23c mimic inhibits the activity of firefly luciferase through the 3'-UTR of METTL3. Moreover, after the miRNA-23c binding site of the 3'-UTR of METTL3 is mutated, miRNA-23c cannot inhibit the activity of firefly luciferase.

[0014] Three, the expression and knockdown of miRNA-23c in EJ cells are detected to detect the change of METTL3 mRNA level. The mRNA expression level of METTL3 is significantly down-regulated after overexpression of miRNA-23c; the mRNA expression level of METTL3 is significantly up-regulated after knockdown of miRNA-23c.

[0015] The miRNA-23c gene in the EJ cell line affects the proliferation and migration of cells, and plays an important role in the occurrence of bladder cancer. METTL3 is a gene related to epithelial-mesenchymal transition, and its expression is related to the occurrence and development of bladder cancer. miRNA-23c inhibits the translation of target gene mRNA or directly degrades target mRNA by complementing the 3'-UTR of the mRNA of the target gene METTL3. In this experiment, bioinformatics analysis and vector construction are used to verify that METTL3 is a target gene of miRNA-23c by luciferase reporter gene analysis in EJ cells, and qRT-PCR technology is used to further verify the finding in EJ cells. This application first reports that METTL3 can be a target gene of miRNA-23c. This application provides a certain application value for using miRNA as a drug target for bladder cancer. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 A columnar statistical chart for qRT-PCR detection of the expression of METTL3 mRNA in bladder cancer cells is shown in the following table:

[0017] Figure 2The results of Western blot detection of METTL3 protein expression in bladder cancer cells are shown in the columnar chart;

[0018] Figure 3 The Western blot image of METTL3 in bladder cancer cells is shown in the Western blot image;

[0019] Figure 4 The columnar chart of the dual-luciferase reporter assay of miRNA-23c and target gene METTL3 3'UTG is shown in the columnar chart;

[0020] Figure 5 The regulatory relationship between miRNA-23c and METTL3 is shown in the graph;

[0021] Figure 6 The schematic diagram of the binding site of miR-23c and target gene METTL3 is shown in the schematic diagram. DETAILED DESCRIPTION

[0022] The application will be further described in detail below with reference to the accompanying drawings and specific examples.

[0023] Unless otherwise specified, the reagents, methods and equipment used in the application are conventional reagents, methods and equipment in the technical field, and the kits and materials used in the following examples are commercially available.

[0024] Example 1: Expression of METTL3 in bladder cancer clinical case cells

[0025] A. The process of RT-qPCR detection of METTL3 mRNA expression in bladder cancer cells is as follows:

[0026] (1) The bladder cancer case tissue sample was lysed by TRIZOL method, and was placed at room temperature for 5 min and mixed thoroughly;

[0027] (2) 200 μL of chloroform was added, and the sample was inverted and shaken by hand for 15 s, and was placed at room temperature for 2-3 min, and was centrifuged at 15000 rpm at 2-8°C for 15 min; it can be seen that the specimen is divided into three layers, and the RNA exists in the upper aqueous phase;

[0028] (3) The supernatant was taken into a 1.5 mL Eppendorf tube, 600 μL of chloroform was added and mixed, and was centrifuged at 15000 rpm for 5 min; the RNA was carefully transferred to another tube, and two times the volume of isopropanol was added to the 1.5 mL Eppendorf tube and mixed evenly; it was placed at -20°C for 10 min, and was centrifuged at 13000 rpm at 4°C for 10 min; after centrifugation, a cotton-like precipitate was seen at the bottom of the tube, which was the RNA precipitate;

[0029] (4) Discard the supernatant, add 500 μL of 75% ethanol, and wash the RNA precipitate by repeatedly pipetting with a pipette. Centrifuge at 8000 rpm at 4°C for 5 min. Discard the supernatant and place the tube opening on the clean bench for 2-3 min to precipitate the dried RNA. Reverse transcribe the RNA into cDNA, and the reverse transcription reaction is performed according to the Vazyme reverse transcription kit instructions.

[0030] The reverse-transcribed sample was subjected to qRT-PCR, and the results are shown in FIG. 2. Figure 1 As can be seen from the figure, the expression of METTL3 mRNA is increased in bladder cancer.

[0031] The PCR instrument is a T100 Thermal Cycler of BIO-RAD Company, and the Applied Biosystems 7500 Real-Time PCR; the reagent is a 5x HiScript II Select qRTSuperMix of Vazyme Company. This reagent contains Taq DNA polymerase, dNTP mix, and SYBR Green dye. U6 is used as an internal reference gene.

[0032] The primers used in this experiment are as follows:

[0033] The METTL3 primer Forward: 5'-ACACTGCTTGGTTGGTGTCA-3', Reverse: 5'-GCGAGTGCCAGGAGATAGTC-3'.

[0034] The process of detecting the expression of METTL3 protein in bladder cancer cells by Western blot is as follows:

[0035] (1) Mix 40 μg of protein sample in the loading buffer and boil at 95-100°C for 5 min;

[0036] (2) Separate the protein in a 12% separation gel and a 5% concentrated gel by polyacrylamide gel electrophoresis; the electrophoresis conditions are as follows: concentrated gel: 80V; 20 min; separation gel: 110V; 70 min;

[0037] (3) After electrophoresis, transfer the protein on the separation gel to the PVDF membrane; the transfer conditions are as follows: 300 mA; 90 min;

[0038] (4) After transfer, block the membrane with blocking solution (20 mL TBST, 1 g skimmed milk powder, 0.3 g BSA) for 2 h, then incubate with the appropriate dilution of primary antibody: anti-METTL3 (1:1000) at 4°C overnight;

[0039] (5) After taking out the membrane in the primary antibody diluent, wash with TBST for three times, each time for 15 min;

[0040] (6) Incubate the membrane with HRP-conjugated secondary antibody (1:5000) at room temperature for 2 h; after the incubation of the secondary antibody is completed, wash the membrane with TBST for three times, each time for 15 min; finally, determine the target protein by using the ECL system, and quantitatively analyze the results of western blot by using Image J software, and the results are shown in Figure 2 and Figure 3 It can be seen from the figure that METTL3 protein is highly expressed in bladder cancer.

[0041] Example Two: Dual-luciferase reporter assay

[0042] Insert the 3'UTR of METTL3 into the 3'UTR end of the reporter plasmid Luciferase, and mutate some sites in the fragment, and also insert the mutated fragment into the 3'UTR end of the reporter plasmid Luciferase, construct WT and MUT Luciferase Reporter Plasmids, and transfect the plasmid into bladder cancer cells with NC mimic, miR-23c mimic, NC inhibitor, and miR-23c inhibitor. 48 h after transfection, add a specific luciferase substrate, and luciferase reacts with the substrate to produce fluorescence. The intensity of fluorescence can be detected to determine the activity of luciferase, and then it can be judged whether miR-23c can interact with METTL3. The results are shown in Figure 4 It can be seen from the figure that miR-23c can target and regulate the expression level of METTL3. The NC mimic, miR-23c mimic, NC inhibitor, and miR-23c inhibitor used in the experiment are purchased from Genepharma Company. The binding site of miR-23c and target gene METTL3 is shown in Figure 6 .

[0043] Example Three: qRT-PCR verification of the inhibitory effect of miR-23c on endogenous METTL3 gene in EJ cells

[0044] EJ cells were evenly spread in 6-well plates, and 24h later, NC mimic, miR-23c mimic, NC inhibitor, miR-23c inhibitor were transfected into bladder cancer cells. After 6h of transfection, the cell culture solution was replaced with fresh culture solution. After 48h of transfection, the cells were collected, RNA was extracted, reverse transcribed into cDNA using a Takara kit, and then qRT-PCR detection was performed. The expression of METTL3 in the reverse-transcribed sample was detected by qRT-PCR, and the results are shown in FIG. 1. Figure 5 As can be seen from the figure, overexpression of miRNA-23c significantly down-regulates the mRNA expression level of METTL3, and knockdown of miRNA-23c significantly up-regulates the mRNA expression level of METTL3, so miRNA-23c has an inhibitory effect on the expression of METTL3.

[0045] In summary, the miRNA disclosed in the present application is complementary to the 3'-UTR of the mRNA of the METTL3 gene of bladder cancer, can inhibit the translation of the target gene mRNA or directly degrade the mRNA of the target gene. The luciferase reporter gene analysis in the EJ cell line verified that METTL3 is the target gene of miRNA-23c, and then overexpression of miRNA-23c in the EJ cell line was found to significantly down-regulate the mRNA level of METTL3. The present application first reports that METTL3 can be a target gene of miRNA-23c, and the present application provides a certain application value for the drug target of bladder cancer treatment using miRNA.

Claims

1. Application of miRNA-23c as a gene targeting METTL3 in preparation of a drug for preventing or treating bladder cancer, wherein the nucleotide sequence of the miRNA-23c is 5'-AUCACAUUGCCAGUGAUUACCC-3'. 2.The use of miRNA-23c as a gene targeting METTL3 in the preparation of a drug for preventing or treating bladder cancer according to claim 1, characterized in that, The drug is used for inhibiting the expression of the METTL3 gene in bladder cancer; or the drug is used for down-regulating the expression level of the METTL3 gene in bladder cancer.

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