CEP55 3’UTR Functions As A CeRNA In Inducing Epithelial-Mesenchymal Transition Of Bladder Cancer Cells Via Regulating Mir-497-5p Activity

Objectives:To investigate whether CEP55 3’UTR could function as a competitive endogenous RNA(ceRNA)to regulate PTHLH and HMGA2 expression and induce epithelial-mesenchymal transition of bladder cancer cells via regulating miR-497-5p activity.Methods:(1)Bioinformatics analysis was performed to screen CEP55 associated ceRNAs.Luciferase report assay and quantitative polymerase chain reaction(qRT-PCR)were employed to determine whether CEP55 3’UTR could function as ceRNA to induce the expression of PTHLH and HMGA2 via regulating miR-497-5p activity.(2)Expression of CEP55,PTHLH,HMGA2 and miR-497-5p were examined using qRT-PCR or western blotting assay in bladder cancer cells and bladder cancer tissues.(3)Wound healing assays,Transwell migration and invasion assays.Cell proliferation and viability assays,Flow-cytometric analysis of apoptosis,and Cell morphology were performed to determine whether CEP55 3’UTR could function as ceRNA to promote the proliferation,migration and invasion of bladder cancer EJ cells and anti-apoptosis via regulating miR-497-5p activity.(4)MTT assays,Flow-cytometric analysis of apoptosis,and Tumor formation assays were performed to investigate the effect of CEP55 3’UTR on cisplatin sensitivity in bladder cancer EJ cells in vitro and in vivo.(5)Western blotting and immunofluorescent assays were used to detect the expression of EMT markers,transcription factors and MAPK pathway related proteins(MEK1/2,Erk1/2,p38,p-MEK1/2,p-Erk1/2 and p-p38)in CEP55 3’UTR overexpression group or depletion group.Results:(1)CEP55 3’UTR、PTHLH 3’UTR and HMGA2 3’UTR were targeted by miR-497-5p.CEP55 3’UTR could function as a ceRNA to increase PTHLH and HMGA2 expression.(2)Result of qRT-PCR and Western blot showed that the expression of CEP55,PTHLH and HMGA2 were frequently up-regulated in bladder cancer cells,while the mRNA level of miR-497-5p was down-regulated in bladder cancer cells.(3)CEP55 3’UTR overexpression or miR-497-5p inhibition significantly promoted bladder cancer EJ cells migration,invasion,proliferation and inhibited its apoptosis,whereas opposite results were obtained when miR-497-5p was overexpressed or the 3’UTR of CEP55 was knocked down by siRNA.However,when si-CEP55 was co-expressed with miR-497-5p inhibitor,its inhibition on cell migration,invasion,proliferation and survival lost.It suggested that there was an opposite effect between miR-497-5p and CEP55 3’UTR.And the effect of silencing CEP55 3’UTR was,at least partly,dependent on the loss of inhibition on miR-497-5p function.(4)CEP55 3’UTR overexpression in EJ cells reduced the sensitivity of tumors to cisplatin in vitro and in vivo.(5)CEP55 3’UTR overexpression significantly increased the Vimentin,Twist and Snail levels compared to the control.In contrast,opposite results were obtained when the 3’-UTR of CEP55 was knocked down by siRNA.(6)CEP55 3’UTR overexpression significantly increased P38,p-P38,MEK1/2,p-MEK1/2,ERK1/2 and p-ERK1/2 levels compared to the control in EJ cells.In contrast,opposite results were obtained when the 3’-UTR of CEP55 was knocked down by siRNA in TCCSUP cells.Conclusion:Our results suggest that CEP55 3’UTR could function as a competitive endogenous RNA(ceRNA)to regulate PTHLH and HMGA2 expression and promote metastasis,invasion,proliferation and anti-apoptotic activity of bladder cancer cells via regulating miR-497-5p activity,thus inducing epithelial-mesenchymal transition of bladder cancer cells.This mechanism may be associated with the activation of the MAPK pathway.