The Mechanism Of Micro RNA-106b-5p Promoting The Proliferation And Migration Of Bladder Cancer By Targeting CDKN1A

Objective: To explore the role of miR-106b-5p in bladder cancer preliminarily by analyzing the TCGA database and using cell function experiments in vitro.Methods: In this study,the sequencing data of miRNAs in bladder cancer tissues and adjacent tissues from the TCGA database were analyzed to identify the differentially expressed miRNAs between bladder cancer tissues and adjacent tissues,and the miRNAs related to the prognosis of bladder cancer patients were analyzed by combining with the clinical data of patients.In addition,we selected miR-106b-5p,which has not been studied in bladder cancer,to preliminarily explore its role in bladder cancer.We transfected miR-106b-5p inhibitor into bladder cancer cells T24 and investigated its effects on the biological behavior of cell proliferation,apoptosis,migration and invasion by CCK-8 kit,EDU kit,flow cytometry,wound healing test and transwell assay.Meanwhile,we predict the potential target genes of miR-106b-5p by bioinformatics method,and verified it by western blot and luciferase reporter gene assay.Results: Based on analyzing bladder cancer related data in the TCGA database,we screen out 473 differentially expressed miRNAs,of which 379 were highly expressed and 94 were low-expressed.Prognostic analysis based on clinical data showed that among the differentially expressed miRNAs,6 differentially expressed miRNAs were significantly correlated with the prognosis of bladder cancer.Among them,miR-106b-5p and miR-141-3p were highly expressed in bladder cancer,and the prognosis of bladder cancer patients with high expression of miR-106b-5p and miR-141-3p was poor,while the patients with low expression was better.Moreover,the expression of miR-99a-5p,let-7c-5p,miR-145-5p and miR-143-3p was low in bladder cancer,and the prognosis of patients with low expression of miR-99a-5p,let-7c-5p,miR-145-5p and miR-143-3p was poor,while the patients with high expression was better.We singled out miR-106b-5p for study because it has been rarely studied in bladder cancer.The results of q RT-PCR showed that the expression of miR-106b-5p was higher in bladder cancer tissues than in adjacent tissues.Meanwhile,compared with normal human bladder epithelial cells(SV-HUC-1),the expression level of miR-106b-5p was higher in bladder cancer cells(T24,J82).The results of CCK-8 kit,EDU kit,flow cytometry,wound healing test and transwell assay showed that the proliferation,migration and invasion abilities of T24 cells decreased after transfection with miR-106b-5p inhibitor,while the apoptosis rate increased.Bioinformatics method was used to predict that CDKN1 A might be the downstream target gene of miR-106b-5p inhibitor,and Western blot showed that the expression of CDKN1 A was significantly increased in bladder cancer T24 cells after transfection with miR-106b-5p inhibitor,The luciferase reporter gene assay was used to verify the hypothesis,The results showed that the luciferase activity of T24 cells co-transfected with miR-106b-5p inhibitor and CDKN1A-WT was significantly increased,while the luciferase activity of T24 cells co-transfected with miR-106b-5p inhibitor and CDKN1A-MUT was not significantly changed.Conclusion: MiR-106b-5p is highly expressed in bladder cancer tissues and cell lines,and down-regulation of miR-106b-5p can inhibit the proliferation,migration and invasion,and promote apoptosis in bladder cancer.These results suggest that miR-106b-5p may be an oncopromoter gene in bladder cancer.In addition,CDKN1 A is a downstream target gene of miR-106b-5p,and miR-106b-5p may play a role in promoting bladder cancer by targeting CDKN1 A.