Research On The Role Of KLF4 In Epithelial-Mesenchymal Transition Of Bladder Cancer

Objective:To study the effect of KLF4 on epithelial-mesenchymal transition of bladder cancer cell and the mechanism of KLF4 in inhibiting EMT,by regulat the expression of KLF4 gene in bladder cancer cell.Methods:1.Chose two different bladder cancer cell lines and detects the expression of KLF4 gene,which with different degree of differentiation.1.1 RT-PCR and Western blot been used to detecting the expression of KLF4 gene in moderately differentiated 5637 and lower differentiated T24 of human bladder cancer cell.2.Effect of overexpression of KLF4 gene on epithelial-mesenchymal transition in bladder cancer.2.1.The T24 and 5637 cells been infected with KLF4 lentivirus control virus,and the experimental cell lines of overexpressing KLF4 and negative control were built.KLF4 mRNA and protein expression were detected by RT-PCR and Western blot,and the infection results were verified.2.2.The mRNA and protein expression levels of EMT-related genes been detected by RT-PCR and WB in 5637 and T24 cells after overexpression of KLF4,and the expression changes of EMT-related genes detected by Immunofluorescence assay in 5637 and T24 cells after transfected with KLF4 lentivirus.2.3.The Transwell assay been used to detects the invasive ability change after lentivirus transfection and overexpression of KLF4 in 5637 and T24 cells.2.4.Western blot been used to detects the effect of expression of β-catenin,c-Myc and MMP9 proteins about the Wnt signaling pathway,and Immunofluorescence assay was used to detect the expression of β-catenin protein after overexpression of KLF4 in 5637 and T24 cells.3.Effect of silenced KLF4 gene on epithelial-mesenchymal transition in bladder cancer.Description: The LV-KLF4 group cells was established as target cell.The LV-KLF4 groupcell was transfected with the KLF4-siRNA fragment and named the siRNA group.The LV-KLF4 group cell was transfected with the KLF4-NC fragment and named the NC group.3.1.RT-PCR and Western blot been used to detects the mRNA and protein changes of KLF4 in siRNA group and NC group after siRNA fragment transfection of LV-KLF4 cells,and the transfection result was verified.3.2.RT-PCR and Western blot been used to detects the mRNA and protein expression of EMT-related genes in siRNA group and NC group after siRNA fragment transfection into LV-KLF4 cells.Immunofluorescence technique been used to detect the expression changes of EMT-related gene proteins.Results:1.1 The expression of KLF4 moderately differentiated 5637 and lower differentiated T24 cells was significantly different.2.1 The expression level of KLF4 in LV-KLF4 group was significantly higher than that in LV-NC group,which detected by RT-PCR and Western blot.The difference was statistically significant.It was confirmed that LV-KLF4 group and LV-NC group were successful established.Transwell results showed that the migration ability of bladder cancer cell in LV-KLF4 group was significantly lower than that in LV-NC group.2.2 The results of RT-PCR and Western blot showed that the expression of E-cadherin was significantly increased in LV-KLF4 group when compared with LV-NC group,while the expression of interstitial markers N-cadherin and Vimentin were significantly decreased.The results of Immunofluorescence showed that the expression of E-cadherin protein in LV-KLF4 group was significantly increased when compared with LV-NC group,and the number of positive cell increased.The fluorescence expression of N-cadherin protein and Vimentin protein were significantly decreased,and the number of positive cells were decreased.2.3 The results of Western blot showed that the expression of total β-catenin protein andβ-catenin protein in the nucleus of LV-KLF4 group decreased to different degrees,and the expression of c-Myc and MMP9 protein were also significant reduce when compared with LV-NC group.Immunofluorescence experiment showed that the fluorescence expression ofβ-catenin protein was decreased in the LV-KLF4 group,the number of positive cell wasdecreased,and the expression in the nucleus was also significantly reduced when compared with LV-NC group.3.1 The expression levels of KLF4 in siRNA group were significantly lower than those in NC group which detected by RT-PCR and Western blot.The difference is statistically significant.It is confirmed that siRNA group and NC group were successfully constructed.3.2 The results of RT-PCR and Western blot showed that the expression of E-cadherin was significantly decreased in the siRNA group,while the expression of N-cadherin and Vimentin was significantly increased when compared with the NC group.The results of Immunofluorescence assay showed that the fluorescence expression of E-cadherin protein was decreased and the number of positive cells was decreased in the siRNA group,the fluorescence expression of N-cadherin protein and Vimentin protein was significantly increased,and the number of positive cells increased when compared with the NC group.Conclusion:1.Overexpression of KLF4 gene inhibits EMT in bladder cancer cell,while silencing of KLF4 gene expression promotes EMT in bladder cancer cell.2.KLF4 gene induced EMT process in bladder cancer cell may be regulated by Wnt/β-catenin signaling pathway.