Study On Expression, Function And Mechanism Of Ezrin Gene In Human Bladder Transitional Cell Carcinoma

Bladder cancer was the most common cancer in urology.Ezrin has been thought that it may play an important role in genesis,development,invasion metastasis of variety tumor. What kind of role ezrin playing was not yet clear in development of BTCC..Experimental study on siRNA of ezrin transfecting BTCC cell lines was not yet reported at home and abroad. The first time we used siRNA to inhibit BTCC cell line T24 to reduced the ezrin gene expression,to study fuction of ezrin in BTCC genesis,development,invasion.The relationship of RhoA and ezrin in BTCC also was studied in this study. Providing foundation of theory which can explore a new way of gene therapy in BTCC.PartⅠThe expression of ezrin and CD44 in bladder transitional cell carcinomaObjectiveTo explore the expression of ezrin and CD44 in bladder transitional cell carcinoma.MethodsImmunohistochemical method was used to assess the expression of ezrin and CD44 in 60 cases of bladder transitional cell carcinoma and 14 cases of control. RT-PCR was used to determine the mRNA of ezrin and CD44 in 40 cases of bladder transitional cell carcinoma of and 10 cases of control.ResultsThe protein expression of ezrin and CD44 were significantly higher in BTCC than that of the controls (P<0.05). The protein expression of ezrin and CD44 were positively correlated with the histological grade and clinical stage of the BTCC(P<0.05). The mRNA expression of ezrin and CD44 were significantly higher in BTCC than that of controls. The mRNA level of ezrin and CD44 were positively correlated with the histological grade and clinical stage of the BTCC(P<0.05).ConclusionBoth expression of ezrin and CD44 were higher in BTCC. The ezrin and CD44 play important role in the development of BTCC. The ezrin can serve as indicators of diagnosis,prognosis and guide treatment and the new target of the BTCC therapy.PartⅡsiRNA on ezrin effect on biological behavior of bladder carcinoma cellObjectiveTo explore the relationship between the ezrin gene expression in BTCC and the biological behavior of strain.MethodsThe siRNA ezrin probe was designed to interfere and reduce the ezrin gene expression in T24 cell. The RT-PCR and western-blot were used to determined the mRNA and protein of ezrin in the cells, which were treated by siRNA ezrin. Cell growth curve,MTT assay,The flow cytometry,cell scratch experiments and cell migration experiments cell adhere experiments were used to test the biological behavior of cells, which were treated by siRNA ezrin.ResultsThe siRNA silencing effect was observed by the treatment of siRNA ezrin. After the siRNA transfection, the mRNA expression of ezrin in experimental group was significantly lower than that of control (P<0.05). The ezrin protein expression was significantly reduced by the transfection (P<0.05).The growth rate of T24 cells, which was treated by siRNA was much slower than the control group, the difference was statistically significant. The apoptosis rate of experimental group was 30.37±8.37%, which was signicantly increased than those of blank control and negative control(P<0.05). The migrating cells was significantly lower than the control group and negative control group. The number of migrating cells in the experimental group was signicantly smaller than those of control (P<0.05). The treatment of siRNA can reduce the cell adhesion capacity.The adhesion rate of the blank control and negative control and experiment group were 72.3±5.1%,70.5±5.7% and 39.2±4.6% respectively. The difference was statistically different (P<0.05).ConclusionThe siRNA can signicantly decreased the mRNA and protein expression of ezrin in T24 cells and cause the different behavioral. The decreased growth rate, adhesion and migrating capacity and increased apoptosis can be observed after the siRNA treatment.PartⅢThe study of the relationship of ezrin and RhoA in BTCCObjectiveTo explore the the relationship of ezrin and RhoA in BTCCMethodWestern-blot was used to determine the protein expression of Ezrin and RhoA and p-RhoA after the treatment of EGF. The protein expression of ezrin and p-RhoA after the treamtment of EGF and Y-27632.ResultsThe p-RhoA protein expression was increased by 70±11%, 250±23%,420±47% and 490±43%, after the treatment of EGF for 10,30,60,90mins. No difference of the RhoA protein expression was observed after the treatment of EGF. The ezrin protein expression was significantly increased by 170±60%,310±73%,750±47% after the treatment of EGF for 8,16,24 hours. The Y-27632 treatment can decreased the effect of EGF on the expression of p-RhoA and ezrin.ConclusionRhoA may regulatd ezrin as a upstream regulatory signal.