LncRNA 00518 Promotes The Development Of Bladder Cancer By Regulating MiR-101/EZH2

Part ? The expression and role of lncRNA 00518 in bladder cancer Objective:To study the expression level of LncRNA00518(lnc00518)in bladder cancer(BCa)cell lines and tissues and its relationship with clinicopathological parameters and prognosis of BCa patients,and analyze the effect of lnc00518 expression on the biological function of BCaMethods:We downloaded RNA-seq data of BCa and normal bladder mucosa in TCGA database,analyzed the differences by Edger function,and performed prognostic analysis by Survival function.Subsequently,we extracted 72 pairs of BCa tissues and matched paracancerous tissues to extract total RNA.Using qRT-PCR to detect the expression level of lnc00518,analyzed the relationship between lnc00518 expression level and clinicopathological parameters and prognosis.EJ and T24 BCa cell lines with small interference and plasmid construction overexpression and low expression of lnc00518 were used to investigate the effect of lnc00518 on the biological function of BCa cells in vitro using CCK-8 experiments,plate cloning experiments,flow cytometric detection,and Transwell invasion migration experiments.In addition,subcutaneous tumorigenesis in nude mice was used to analyze the effect of lnc00518 on the development of BCa in vivoResults:TCGA database analysis found that the expression of lnc00518 in BCa tissue was higher compared with normal bladder mucosa.The survival analysis demonstrated that the expression of lnc00518 was negatively correlated with the overall survival time of the patients.The subsequent validation of 72 pairs of BCa tissues and matched paracancerous tissues was consistent with the results of TCGA analysis,and lnc00518 was significantly higher in BCa.Analysis of clinical data revealed that the high expression of lnc00518 was associated with TNM stage and histological grade in patients with tumors.Meanwhile,the overall survival rate of patients with high expression of lnc00518 was lower.Comparing with the control group,the proliferation and colony forming ability of the lnc00518-overexpressing cells were significantly increased,the cell cycle G0/G1 phase ratio was reduced,the ability of invasion and migration were significantly enhanced,while knocking-down the lnc00518 expression,the result was reversed.Tumor formation experiments in nude mice showed that tumor growth was slow after knocking down lnc00518,and the final volume and weight were lower Compared with the control group.Conclusion:The expression of lnc00518 in BCa was significantly increased,and the expression level of lnc00518 was positively correlated with TNM stage,histological grade and poor prognosis of BCa.In vitro and in vivo experiments showed that lnc00518 can promote the proliferation,invasion,migration and subcutaneous tumorigenicity of BCa cells.Part ?Effects of lncRNA 00518 on the cell proliferation in bladder cancer through regulating miR-101/EZH2Objective:To search for and validate lnc00518 targeted miRNAs and downstream pathways to explore the specific mechanisms by which lnc00518 promotes BCa proliferation.Methods:By searching the on-line bioinformatics database(RegRNA),the possible miRNA targets of lnc00518 were predicted,and RNA levels of possible target miRNAs were verified and further confirmed by luciferase reporter gene,RIP experiments and recovery experiments.Results:Bioinformatics database analysis revealed potential binding sites for miR-24,miR-100,miR-101,miR-128,and miR-204 with lnc00518.Further validation analysis revealed that knockdown of lnc00518 significantly increased the expression of miR-101,so we chose miR-101 as the targeted miRNA for lnc00518.The qRT-PCR results of BCa tissues and cells showed that the expression of lnc00518 was negatively correlated with miR-101.The results of luciferase reporter and RIP indicated that lnc00518 can directly bind to miR-101.In addition,bioinformatics analysis and luciferase reporter gene results indicate that EZH2 is a miR-101 target-regulated gene.The results of the recovery experiment showed that inhibition of miR-101 restored the down-regulation of lnc00518 on the proliferation,invasion and migration of BCa.Conclusion:Lnc00518 can promote the proliferation,invasion and migration of BCa by up-regulating its target gene EZH2 through competitive binding to miR-101.