The Roles And Mechanisms Of YAP1 In Regulating Bladder Cancer EMT

Bladder cancer(BC)is the most common malignant neoplasm of urological organs,most of which is urothelial carcinoma.Along with the continuous improvement in the technology of transurethral resection and radical resection of bladder cancer,some progress in the popularization of postoperative intravesical chemotherapy has been made;the treatment of bladder cancer has been continuously improved.However,after regular treatment,the primary bladder cancer is still easy to relapse and metastasis,and even lead to death.Therefore,the treatment of bladder cancer is still a worldwide problem.Through studing the related molecular mechanism of YAP1 on the impact of bladder cancer cell epithelial mesenchymal transition will help us to find the clinical methods and strategies for prevention of bladder cancer recurrence,metastasis and progress,and provide new ideas for the treatment of bladder cancer in the future;and provide new therapeutic targets in molecular targeted therapy for bladder cancer.Chapter 1 The expression of YAP1,E-cadherin,and Vimentin in BC tissues,cells and the related clinical significanceObjectives:To detecte the YAP 1 mRNA and protein level in BC,normal tissues and the BC cell lines.Meanwhile,the correlation between the expression of YAP1 or E-cadherin and the clinicopathological features,prognosis was analysed.Methods:From September 2014 to September 2015 in Nanfang Hospital,we collected 18 cases BC sample and 18 fresh normal tissues.Meanwhile,tissue microarray(including 55 BC sample 10 para-carcinoma tissue)were used to test the YAP1,Vimentin and E-cadherin expression by quantitative real-time PCR,western blot and immunohistochemistry,which were diagnosed,staged and graded accord WHO criteria.The correlation between the expression profile of YAP1,E-cadherin and the clinicopathological features,prognosis was analyzed.Results:A significantly higher YAP1 and Vimentin mRNA and protein expression in bladder cancer tissue and cells(P<0.05).The immunohistochemical detection showed YAP1 and Vimentin protein expression in cancer tissue is significantly higher than non cancerous tissue(P<0.05).E-cadherin protein expression in cancer tissue is significantly lower than non cancerous tissue.The correlation analysis showed a positive relation between the YAP1 expression and malignancy,prognosis of tumor,the Vimentin expression.And there was a negative relation between the YAP1 expression and the E-cadherin expressionConclusion:The YAP1 and Vimentin mRNA and protein expression level in bladder cancer tissue were significantly higher than that of non cancerous tissue,E-cadherin mRNA and protein expression level in bladder cancer tissue was significantly lower than that of non cancerous tissue.Carcinoma tissue with higher YAP1 expression level is associated with the malignancy,T staging and prognosis of tumors.Chapter 2 The effects of YAP1 on invasion and EMT in BC in vitro studyObjectives:We constructed the BC cell line UMUC3 and T24 cell with YAP1 knockdown by siRNA and explored the roles of YAP1 on the influence of the invasion and EMT.Methods:The siRNA were used to transfected the UMUC3 and T24 cells respectively.Cell invasion was detected by transwell method.Furthermore,we also explored the related proteins level in EMT by RT-qPCR and western blot.The data was presented as mean ± standard deviation.The one way ANOVA analysis was applied in the comparison among groups.Results:After silencing YAP1 expression with siRNA,the cell invasion capacity was inhibited(P<0.05).Western blot results show the EMT related proteins Vimentin protein expresion were down-regulated and E-cadherin upregulation after YAP1 siRNA.Conclusion:YAP1 plays roles on the invasion,and closely related with bladder cancer cells EMT.Chapter 3 The roles of YAP1 in regulating EMT and the feedback effect of E-cadherinObjective:To investigate the effects of YAP1 on EMT expression in BC cell and to explore the related mechanisms of YAP 1 in BC EMT.Method:Firstly,knockdown of YAP1 expression in UMUC3 and T24 cells by siRNA.RT qPCR was used to detect the ZEB1,Snail 1 expression.Secondly,up-regulate expression of YAP 1 in T24 cell by pcDNA-YAP1.Detect the expression of EMT related proteins.Thirdly,AKT inhibitor was used to block the Akt signaling pathway.Western blot was used to detect the expression of EMT related proteins.Lastly,knockdown of E-cadherin expression in UMUC3 and T24 cell by siRNA.Western blot and RT qPCR were used to detect the YAP1,vimentin,ZEB1,Snail 1 protein expression.The data was presented as mean ± standard deviation.Results:Snail 1 and ZEB1 mRNA expresion were upregulation after silence of YAP1.On the contrary,YAP1,Vimentin,Snail1 and ZEB1 expression could be enhanced after silence of E-cadherin.The effect of YAP1 induced the EMT could partly be blocked by AKT inhibitor.Conclusions:YAP1 plays roles on the effects of EMT through Akt signaling pathway in bladder cancer cells,down-regulated E-cadherin enhanced the effect of EMT,creating a positive feedback loop.