Transforming Growth Factor ?1 Promotes Epithelial-Mesenchymal Transition And Stemness Maintenance Of Bladder Cancer Cells Through Long Non-Coding RNAs

Objective:To explore the role of transforming growth factor ?1 in urothelial bladder cancer,especially in the aspect of epithelial-mesenchymal transition,migration/invasion and cancer sternness.To explore the regulative function of long non-conding RNAs in TGF?1 signaling pathway for presenting a novel diagnostic and therapeutic strategy.Methods:5nM human recombinant TGF?1 and/or 20nM TGF?R1 inhibitor(SB-431542)was added into bladder cancer cell lines-5637,T24 and J82.The morphological features of bladder cancer cell lines under TGF?1 treatment were observed using inverted fluorescence microscope.Epithelial and mesenchymal markers and EMT associated transcriptional factors were detected by quantitive real time PCR and immunoblotting.The migration and invasion capabilities of bladder cancer cell lines were detected by wound healing assay and transwell assay.The ability of bladder cancer cell stemness was detected by flow cytometer for ALDH1 and CD44 positive cell rate and sphere assay.Human lncRNA Discover PCR array was used for lncRNA profiling,which associated with EMT or cancer stemness Dual-luciferase reporter assay was used to analyse the relationship between lncRNAs and TGF? signaling pathway.RNA interfering and plasmids overexpression were used for functional research of lncRNAs.The correlation analysis was performed between lncRNAs and TGF?1 in human baldder cancer specimens.Results:TGF?1 phosphorylated SMAD2 and activated TGF? signaling pathway in 5637,T24 and J82.Treatment of TGF?1 strikingly induced morphological changes of EMT in all of three cancer cells.The epithealial marker(E-cadherin)was downregulated by TGF?1,while the mesenchymal markers(Vimentin,Fibronectin)and the EMT-associated transcriptional factors(SNAI1,SNAI2,ZEB1,ZEB2,TWIST 1)were upregulated.Interestingly,lncRNA-ZEB2NAT was demonstrated to be essential for this TGF?1-dependent process.ZEB2NAT depletion reversed TGF?1-induced EMT and invasion of cancer cells as well as reduced the ZEB2 protein level.Consistently,TGF?1 mRNA expression is positively correlated with ZEB2NAT transcript and ZEB2 protein levels in human bladder cancer specimens.TGF?1 also can induce stemness maintenance through lncRNA-LET.Dual-luciferase reporter assay showed that SMAD4 could bind to the promter and repress the expression of lncRNA-LET.LncRNA-LET depletion promoted stemness maintenance of baldder cancer cells,while lncRNA-LET overexpression reduced.LncRNA-LET reduced NF90 protein stability which affected the sternness change induced by lncRNA-LET.Conclusion:TGF?1 plays an essential role in bladder cancer progress.TGF?1 promotes epithelial-mesenchymal transition and stemness maintenance of bladder cancer cells through lncRNA-ZEB2NAT and lncRNA-LET,respectively.Targeting these paracrine signaling may present a novel therapeutic strategy for bladder cancer.