Application of miRNA-23c as gene targeting METTL3 in preparation of medicine for preventing or treating bladder cancer

Abstract

The invention discloses application of miRNA-23c serving as a gene targeting METTL3 to preparation of a medicine for preventing or treating bladder cancer. The invention discloses miRNA (micro Ribonucleic Acid) for inhibiting the growth of bladder cancer, miRNA-23c is complementary with 3 '-UTR (Untranslated Region) of mRNA (Messenger Ribonucleic Acid) of an METTL3 gene, so that the translation of the mRNA of the target gene can be inhibited or the mRNA of the target gene can be directly degraded. In an EJ cell line, luciferase reporter gene analysis verifies that METTL3 is a target gene of miRNA-23c, then miRNA-23c is over-expressed in the EJ cell line, and it is found that miRNA-23c significantly down-regulates the mRNA level of METTL3. The invention reports that METTL3 can be used as a target gene of miRNA-23c for the first time. The miRNA provided by the invention provides a certain application value in the aspect of drug targets for bladder cancer treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of miRNA-23c as a gene targeting METTL3 in the preparation of a drug for preventing or treating bladder cancer. Background Art

[0002] MicroRNA (miRNA) plays an important regulatory role in the growth, development and occurrence of various diseases of organisms, and has become a hot topic in current life science research. MiRNA is a type of non-coding single-stranded small molecule RNA with a length of 21-22nt. It degrades the target mRNA or inhibits its translation by complementary pairing with the target gene mRNA. Studies have shown that miRNA plays an important role in the occurrence of tumors, and miRNA is likely to become a new approach for cancer treatment and diagnosis. There is a one-to-many relationship between miRNA and the non-coding sequence (3′-UTR or 5′-UTR) of the target gene mRNA. The phenomenon of dysregulated expression of miRNA groups is often found in cancer cases, and changes in the expression of these miRNAs are closely related to the occurrence of cancer. In in vitro experiments, changing the expression of one or more miRNAs can promote or inhibit the deterioration of cancer cells. MiRNAs that promote the deterioration of cancer cells can be considered as a type of oncogene, and vice versa.

[0003] Bladder cancer (BC) ranks first in the incidence of urogenital tumors in my country, and its incidence rate is second only to prostate cancer in Western countries. According to statistics, bladder cancer ranked sixth in the number of new male cases in the world in 2020, with about 440,000 cases. Surgical resection and chemotherapy are the two mainstream treatment strategies for bladder cancer. However, most bladder cancer patients are diagnosed in the middle and late stages once they are discovered. Even patients who receive active treatment still face the dilemma of recurrence, poor prognosis, and low survival rate. Therefore, it is particularly urgent to deeply explore the molecular regulatory mechanism of the occurrence and development of bladder cancer in order to provide new intervention targets 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 modifications, and has the functions of regulating RNA splicing, localization, transport, stability, and translation efficiency. The METTL3 catalytic process modifies most m6A sites and is considered to be the most common m6A pathway. METTL3-mediated m6A modification is also a key promoter of EMT and metastasis of malignant tumors. miRNA may become a new marker for early diagnosis and progression of bladder cancer, which will contribute to the accurate diagnosis and personalized treatment of bladder cancer. Summary of the invention

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

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] Provided is the use 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 to inhibit the expression of METTL3 gene in bladder cancer; or the drug is used to down-regulate the expression level of METTL3 gene in bladder cancer.

[0008] On the other hand, the present invention also provides a pharmaceutical preparation for preventing or treating bladder cancer, comprising (1) an effective amount of miRNA-23c as an active ingredient, and (2) a selective pharmaceutically acceptable carrier.

[0009] Preferably, the pharmaceutical dosage form is capsule, tablet, powder, oral solution, pill, tincture, syrup or injection.

[0010] The research ideas of the present invention are further described as follows:

[0011] The present invention uses 30 clinical samples of bladder cancer and qRT-PCR to detect the gene expression of the samples, and finds that the expression of a gene related to epithelial-mesenchymal transition is related to the occurrence and development of bladder cancer. METTL3 is upregulated in bladder cancer cells at both the mRNA and protein levels. Differentially expressed miRNAs were screened through TCGA bladder cancer data, and the differential miRNAs of METTL3 were predicted by the multiMiR R software package. The miRNAs upstream of METTL3 were then predicted by the Starbase database. The intersection of the three was used to find miRNAs that may mediate the participation of METTL3 in the regulation of bladder cancer, and it was found that miRNA-23c can target METTL3. The present invention confirms that miRNA-23c can target and inhibit the expression of the METTL3 gene through luciferase activity detection experiments and overexpression analysis of miRNA-23c.

[0012] The total RNA extracted from cells by Trizol reagent from a biological company was reverse transcribed to detect the expression of METTL3 in clinical cases of bladder cancer. The study found that METTL3 was significantly increased in bladder cancer compared with adjacent tissues of the cases.

[0013] Second, according to bioinformatics prediction, the 3'UTR of METTL3 was inserted into the 3'UTR end of the reporter plasmid Luciferase, and some sites of the fragment were mutated. The mutated fragment was also inserted into the 3'UTR end of the reporter plasmid Luciferase to construct WT and MUT Luciferase Reporter Plasmids. The plasmids were transfected into bladder cancer cells with NC mimic, miRNA-23cmimic, NC inhibitor, and miRNA-23c inhibitor. 48 hours after transfection, a specific luciferase substrate was added, and the luciferase reacted with the substrate to produce fluorescence. The activity of luciferase can be determined by detecting the intensity of fluorescence, and then it can be determined whether miRNA-23c can interact with METTL3. It shows that miRNA-23c mimic inhibits the activity of firefly luciferase through the 3'-UTR of METTL3. Moreover, after the point mutation of the miRNA-23c binding site in the 3'-UTR of METTL3, miRNA-23c cannot inhibit the activity of firefly luciferase.

[0014] Third, miRNA-23c was overexpressed and knocked down in EJ cells to detect the changes in METTL3 mRNA level. Overexpression of miRNA-23c significantly downregulated the mRNA expression level of METTL3; knocking down miRNA-23c significantly upregulated the mRNA expression level of METTL3.

[0015] The miRNA-23c gene in the EJ cell line affects cell proliferation, migration, etc., 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 its target gene METTL3 mRNA. This experiment used bioinformatics analysis and vector construction, and then verified METTL3 as a target gene of miRNA-23c using luciferase reporter gene analysis in EJ cells, and further verified the finding using qRT-PCR technology in EJ cells. This invention reports for the first time that METTL3 can be used as a target gene of miRNA-23c. This invention provides certain application value in using miRNA to provide drug targets for bladder cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a bar chart showing the expression results of METTL3 mRNA in bladder cancer cells detected by qRT-PCR;

[0017] Figure 2This is a bar chart showing the expression of METTL3 protein in bladder cancer cells detected by Western blot;

[0018] Figure 3 Western blot image of METTL3 in bladder cancer cells;

[0019] Figure 4 This is a bar graph of the dual luciferase reporter assay analysis of miRNA-23c and the target gene METTL3 3′UTG;

[0020] Figure 5 This is a diagram of the regulatory relationship between miRNA-23c and METTL3;

[0021] Figure 6 Schematic diagram of the binding site between miR-23c and the target gene METTL3. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

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

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

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

[0026] (1) Bladder cancer tissue samples were lysed using the TRIZOL method, left to stand at room temperature for 5 min, and mixed thoroughly;

[0027] (2) Add 200 μL of chloroform, shake by hand for 15 seconds, let stand at room temperature for 2-3 minutes, and centrifuge at 15,000 rpm for 15 minutes at 2-8°C. It can be seen that the specimen is divided into three layers, and RNA is present in the upper aqueous phase.

[0028] (3) Transfer the supernatant to a 1.5 mL Eppendorf tube, add 600 μL chloroform and mix well, centrifuge at 15,000 rpm for 5 min; carefully transfer the RNA to another tube, add twice the volume of isopropanol to the 1.5 mL Eppendorf tube and mix well; place at -20°C for 10 min, centrifuge at 13,000 rpm and 4°C for 10 min, and cotton-like precipitate can be seen at the bottom of the tube after centrifugation, which is the RNA precipitate;

[0029] (4) Discard the supernatant, add 500 μL 75% ethanol, use a pipette to repeatedly blow and wash the RNA precipitate, and centrifuge at 8000 rpm and 4°C for 5 min; discard the supernatant and place the tube opening in an ultra-clean bench for 2-3 min to precipitate and dry the RNA; reverse transcribe the RNA into cDNA, and perform the reverse transcription reaction according to the instructions of the Vazyme reverse transcription kit.

[0030] The reverse transcribed samples were subjected to qRT-PCR, and the results were as follows: Figure 1 As shown in the figure, it can be seen that the expression of METTL3 mRNA is increased in bladder cancer.

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

[0032] The primers used in this experiment are:

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

[0034] B. The process of Western blot detection of METTL3 protein expression in bladder cancer cells is as follows:

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

[0036] (2) Protein separation was performed by polyacrylamide gel electrophoresis with 12% separation gel and 5% stacking gel; the electrophoresis conditions were: stacking gel: 80V; 20min; separation gel: 110V; 70min;

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

[0038] (4) After transfer, the membrane was blocked with blocking solution (20 mL TBST, 1 g skim milk powder, 0.3 g BSA) for 2 h, and then incubated with an appropriately diluted primary antibody: anti-METTL3 (1:1000) at 4 °C overnight;

[0039] (5) After removing the membrane from the primary antibody dilution solution, wash it three times with TBST, each wash for 15 minutes;

[0040] (6) The membrane was incubated with HRP-conjugated secondary antibody (1:5000) at room temperature for 2 h. After the secondary antibody incubation, the membrane was washed three times with TBST, each time for 15 min. Finally, the target protein was detected using the ECL system, and the Western blot results were quantitatively analyzed using Image J software. The results are shown in Figure 2. Figure 2 and Figure 3 As shown in the figure, it can be seen that METTL3 protein is highly expressed in bladder cancer.

[0041] Example 2: Dual luciferase reporter gene analysis

[0042] The 3'UTR of METTL3 was inserted into the 3'UTR end of the reporter plasmid Luciferase, and certain sites of the fragment were mutated. The mutated fragment was also inserted into the 3'UTR end of the reporter plasmid Luciferase to construct WT and MUTLuciferase Reporter Plasmids. The plasmids and NC mimic, miR-23cmimic, NC inhibitor, and miR-23c inhibitor were transfected into bladder cancer cells. 48 hours after transfection, a specific luciferase substrate was added, and the luciferase reacted with the substrate to produce fluorescence. The activity of luciferase can be determined by detecting the intensity of the fluorescence, and then it can be determined whether miR-23c can interact with METTL3. The results are as follows: Figure 4 As shown in the figure, it can be seen 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 were purchased from Genepharma. The schematic process of the binding site of miR-23c and the target gene METTL3 is shown in Figure 6 shown.

[0043] Example 3: qRT-PCR verification of the inhibitory effect of miR-23c on the endogenous METTL3 gene in EJ cells

[0044] EJ cells were evenly plated in a 6-well plate. After 24 hours, NC mimic, miR-23c mimic, NC inhibitor, and miR-23c inhibitor were transfected into bladder cancer cells. After 6 hours of transfection, the cell culture medium was replaced with fresh culture medium. After 48 hours of transfection, cells were collected, RNA was extracted, and reverse transcribed into cDNA using a Takara kit, followed by qRT-PCR detection. The reverse transcribed samples were used to detect the expression of METTL3 by qRT-PCR. The results are shown in Figure 2. Figure 5 As shown in the figure, it can be seen that overexpression of miRNA-23c significantly downregulated the mRNA expression level of METTL3; knockdown of miRNA-23c significantly upregulated the mRNA expression level of METTL3; thus, it can be seen that miRNA-23c has an inhibitory effect on the expression of METTL3.

[0045] In summary, the present invention discloses a miRNA that inhibits the growth of bladder cancer, and miRNA-23c is complementary to the 3′-UTR of the METTL3 gene mRNA, which can inhibit the translation of the target gene mRNA or directly degrade the target gene mRNA. Luciferase reporter gene analysis was used in the EJ cell line to verify that METTL3 is the target gene of miRNA-23c. Then, miRNA-23c was overexpressed in the EJ cell line, and it was found that miRNA-23c significantly downregulated the mRNA level of METTL3. The present invention reports for the first time that METTL3 can be used as a target gene of miRNA-23c. The invention provides certain application value in terms of using miRNA as a drug target for the treatment of bladder cancer.

Claims

1. Application of miRNA-23c as a gene targeting METTL3 in the preparation of drugs for preventing or treating bladder cancer.

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 nucleotide sequence of the miRNA-23c is 5'-AUCACAUUGCCAGUGAUUACCC-3'.

3. Use of miRNA-23c according to claim 1 or 2 as a gene targeting METTL3 in the preparation of a drug for preventing or treating bladder cancer, characterized in that: The drug is used to inhibit the expression of the METTL3 gene in bladder cancer; or the drug is used to down-regulate the expression level of the METTL3 gene in bladder cancer.

4. A pharmaceutical preparation for preventing or treating bladder cancer, characterized in that: The invention comprises (1) an effective amount of miRNA-23c as an active ingredient, and (2) a selective pharmaceutically acceptable carrier.

5. A pharmaceutical preparation for preventing or treating bladder cancer according to claim 4, characterized in that: The drug dosage form is capsule, tablet, powder, oral liquid, pill, tincture, syrup or injection.

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