Transforming Growth Factor β1 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 β1 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 TGFβ1 signaling pathway for presenting a novel diagnostic and therapeutic strategy.Methods:5nM human recombinant TGFβ1 and/or 20nM TGFβR1 inhibitor(SB-431542)was added into bladder cancer cell lines-5637,T24 and J82.The morphological features of bladder cancer cell lines under TGFβ1 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 TGFβ signaling pathway.RNA interfering and plasmids overexpression were used for functional research of lncRNAs.The correlation analysis was performed between lncRNAs and TGFβ1 in human baldder cancer specimens.Results:TGFβ1 phosphorylated SMAD2 and activated TGFβ signaling pathway in 5637,T24 and J82.Treatment of TGFβ1 strikingly induced morphological changes of EMT in all of three cancer cells.The epithealial marker(E-cadherin)was downregulated by TGFβ1,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 TGFβ1-dependent process.ZEB2NAT depletion reversed TGFβ1-induced EMT and invasion of cancer cells as well as reduced the ZEB2 protein level.Consistently,TGFβ1 mRNA expression is positively correlated with ZEB2NAT transcript and ZEB2 protein levels in human bladder cancer specimens.TGFβ1 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:TGFβ1 plays an essential role in bladder cancer progress.TGFβ1 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.