LncRNAH19 Regulates Epithelial–Mesenchymal Transition And Metastasis Of Bladder Cancer By MiR-29b-3p As Competing Endogenous RNA

ObjectiveTo obtain the reliable evidence of lnc RNA H19 could competitively bind to miR-29 as a ce RNA to relieve micro RNA repression for target gene DNMT3 B and regulate factors related to EMT and EMT signaling pathways,which promote the EMT and metastasis of bladder cancer cells.To elucidate the function of lnc RNA H19,reveal the new molecular mechanism of EMT and invasion and metastasis of bladder cancer,and provide the original scientific basis for finding target genes and drugs for diagnosis and treatment of bladder cancer.MethodsHere,we screened lnc RNA and m RNA expression profiles of bladder cancer(BC)with microarray assay.The expression of H19 and DNMT3 B in human bladder cancer and normal bladder was verified by qRT-PCR.To detect the effects of H19 on proliferation,motility and invasion of bladder cancer cells,CCK8,Ed U,formation of colonies assays,wound healing assay,transwell assay and were conducted when H19 was down-or up-regulated.we further probed the role of H19 and the relationship between H19 and DNMT3 B during BC progression with xenograft models.To evaluate the influences of H19 expression on EMT marker proteins and DNMT3 B levels,Immunofluorescence and Immunohistochemistry probes were performed in vivo.Then the binding sites of H19 and DNMT3 B and miR-29b-3p were predicted by the bioinformatic website.H19 could act as a ce RNA through harboring miR-29b-3p,and hence functionally abolished the endogenous suppressive effect on target gene for binding to the 3′ UTR of DNMT3 B.Subsequently,we analyzed the intracellular localization and expressions of H19 and miR-29b-3p by FISH in human bladder cancer tissue,carcinoma adjacent tissue and BC T24 cells.To prove the endogenous binding between miR-29b-3p and H19 at cellular level,a RNA immunoprecipitation(RIP)and RNA pull down were executed to detect miR-29b-3p or H19 by q PCR assay.To testify the regulated relationship between H19,DNMT3 B and mir-29b-3p,we tested whether H19 was able to regulate target DNMT3 B of miR-29b-3p by qRT-PCR and Western blot in BC cells.Subsequently,we explored whether H19 could affect EMT marker proteins’ levels by regulating DNMT3 B in vitro through WB assay.ResultsH19 and DNMT3 B expressions are upregulated in bladder cancer tissues and cells were executed by microarray Assayes and verfied by RT-q PCR.CCK-8,Ed U,formation of colonies,wound healing,transwell assays and cytoskeleton showed that up-regulating H19 significantly facilitated proliferation,migration and invasion,mobility of bladder cancer cells,accordingly,si H19 displayed the opposite effects.H19 promotes tumorigenesis,metastasis and angiogenesis of bladder cells in vivo.H19 regulates the expressions of proteins associated with EMT and DNMT3 B protein levels are observed in vivo.Bioinformatic prediction showed that both H19 and DNMT3 B could be bind to miR-29b-3p.A dual-luciferase reporter assay initially suggested that the miR-29b-3p and H19 might combine with each other.FISH assay revealed that H19 and miR-29b-3p has a co-localization in the BC tissues and cells.The endogenous combination between miR-29b-3p and H19 was further confirmed using affinity pull-down of miR-29b-3p by biotin-labeled H19(RNA pull down)and RIP assay in vitro.We verified that the regulated miR-29b-3p could reduce the expression of DNMT3 B with qRT-PCR and WB in BC cells,and the expression changes of H19 and DNMT3 B were consistent.Finally,WB experiments showed that H19 regulates the expression of DNMT3 B by interacting with miR-29 b,which affects the EMT process.Conclusion1.qRT-PCR analysis revealed H19 and DNMT3 B were markedly increased in BC tissues and the expressions of H19 and DNMT3 B were positive correlated.2.H19 affects proliferation,migration,invasion,mobility,angiogenesis and EMT process of bladder cancer cells in vivo and vitro.3.H19 regulates expressions of the miR-29 b target gene DNMT3 B and EMT-associated proteins by binding to miR-29b-3p.