The Anti-tumor Role And Mechanism Of MiR-501-3p On Renal Cell Carcinoma

Background:Renal cell carcinoma(RCC)is a common malignant tumor in the urinary system originated from renal parenchymal urothelial system.It accounts for 80%～90%of the malignant tumors of kidney and the incidence rate is about 2.1%among all kinds of tumors in human.Recently,the incidence rate of RCC has been increasing due to smoking and diet changes.RCC is easy to progress with poor prognosis,but yet its molecular mechanism is not clear.To elucidate the biological mechanism of RCC is of great clinical significance for early diagnosis and treatment of RCCMicroRNA(miRNA)is a kind of noncoding RNA encoded by endogenous genes.It regulates gene expression through targeting the 3’-UTR of targeted gene mRNA.Studies show that miRNA such as miR-501-3p is involved in tumor development.miR-501-3p overexpression can inhibit the proliferation and clone formation of prostate cancer cells and induce G1 phase arrest.miR-501-3p also plays an inhibitor or promoter role in lung cancer,colorectal cancer,pancreatic duct cancer and other tumors.However,the expression pattern and biological function of miR-501-3p in RCC are unclear,and effects of miR-501-3p acting on the proliferation and migration of RCC and its molecular mechanism are unknownObjection:Analyze the expression level of miR-501-3p in RCC and the epigenetic mechanism by bioinformatics methods.Investigate the biological function of miR-501-3p in RCC by functional experiments.Verify the target gene of miR-501-3p by bioinformatics prediction and dual-luciferase reporter gene assay.Investigate the biological function of the target gene in RCC,verify the regulatory mechanism of miR-501-3p and the potential downstream molecular pathway.Method:1.Analyze the expression pattern of miR-501-3p in RCC by bioinformatics method and investigate whether down-regulation of miR-501-3p is related to the abnormal methylation of CpG island in the promoter region.2.Up regulate miR-501-3p by transfecting miR-501-3p mimic into 786-O and Caki-1 cells.Investigate the effects of miR-501-3p acting on the proliferation and migration of RCC cells by CCK-8,flow cytometry,colony formation assay and Transwell migration experiment.3.Verify the direct target gene of miR-501-3p in RCC by bioinformatics prediction and dual-luciferase reporter gene assay.Explore the effect of target gene on the proliferation and migration of RCC cells by CCK-8 experiment,flow cytometry,colony formation assay and Transwell migration experiment.4.Explore the regulatory mechanism of miR-501-3p and target gene through rescue experiment and m6ADot Blot Assay.Result:1.We find that the expression of miR-501-3p in RCC is significantly lower than that in normal tissues.However,there is no CpG island in the region of about 5000 bp before and after transcription start site of miR-501-3p.miR-501-3p can inhibit RCC development,and its low expression in RCC has nothing to do with the abnormal methylation of promoter region.2.Up-regulation of miR-501-3p can significantly inhibit the proliferation,clone formation and migration of RCC cells,but induce G1 phase arrest of RCC cells3.We confirm that WTAP is the target gene of miR-501-3p in RCC by bioinformatics prediction and dual-luciferase reporter gene assay4.WTAP expression is significantly higher in RCC cells than that in normal tissues WTAP knockdown can significantly inhibit the proliferation,clone formation and migration of RCC cells and induce G1 phase arrest.WTAP regulates the cell cycle of RCC through CDK25.The rescue experiment confirms that miR-501-3p overexpression could effectively inhibit the proliferation and migration of RCC cells induced by WTAP.m6A Dot Blot Assay confirms that WTAP knockdown or miR-501-3p overexpression could down-regulate the expression of m6A in RCC cells.However,there is no m6A modification site on CDK2 mRNA,so we speculate that m6A modification is not the mechanism of miR-501-3p/WTAP/CDK2 axis in renal cancer.Conclusion:Up-regulation of miR-501-3p can inhibit the proliferation and clone formation of RCC cells by inducing G1 phase arrest.WTAP is the direct target gene of miR-501-3p in RCC.miR-501-3p/WTAP/CDK2 regulate tumor progression,which proposing a new theory for the pathogenesis of renal cancer.