Effect On The Epithelial-to-mesenchymal Transition Through Knockdown Of FoxM1 Gene In T24 Cell Line

Objective: The study choosed T24 bladder cancer cell line as the research object to observe the influence of knockdown of transcription factor FoxM1 on Epithelial-to-Mesenchymal transition(EMT) of hunman bladder cancer cell line T24 and to preliminary explored the mechanism of EMT in bladder cancer cells.Methods: Interference plasmid FoxM1-shRNA was constructed, then transfected into T24 cells by lipofectamine2000. The expression mRNA and protein of FoxM1 were detected by reverse transcription-polymerase chain reaction(RT-PCR) and western blot after transfection 48 h and the most significant interference group was selected to carry out the formal experiment. FoxM1-shRNA and NC-shRNA were transfected into T24 cells to set up two groups as follows: shRNA-FoxM1-T24 and shRNA-NC-T24.ShRNA-FoxM1-T24 was as the experimental group, shRNA-NC-T24 and T24 cells without transfection as the control group. After 48 h changes in cell morphology were observed by microscope. Then the expressions of FoxM1 and epithelial mesenchymal markers E-cadherin、Vimentin were detected by RT-PCR and western blot in all three groups. The effect of down-regulation of FoxM1 was observed in T24 cell line.Results: 1.RT-PCR and western-blot results showed that FoxM1 was over expressed in T24 cells. Relative to the control group, the expression mRNA and proteininof FoxM1 in shRNA transfection group apparently decreased(P<0.01), the interference efficiency of shRNA3 was obviously higher than other three positive interference carriers(shRNA1, shRNA2, shRNA4). So interference carrier shRNA3 was used for subsequent experiment.2.Compared with control group and negative control group, the expression mRNA and protein of FoxM1 in shRNA3 transfection group apparently decreased(P<0.01), suggesting that FoxM1 gene was knocked down in T24 cells. Under the high power microscope, the cell in negative control group and control group became polygon and lost cell polarity with larger intercellular space,isolating cell, mesenchymal phenotype cell.However, T24 cells most appeared oval and connected tightly, transforming towards normal epithelial phenotype cell 48 hours after transfected FoxM1-shRNA3.3.While the expression of mesenchymal marker Vimentin decreased obviously, the expression of epithelial marker E-cadherin increased remarkably after 48 hours transfected FoxM1-shRNA3, the difference was statistically significant relative to control group(P<0.01). The experiment result showed that the down-regulation of FoxM1 gene expression induced T24 cells changing from mesenchymal phenotype cell to normal epithelial phenotype cell.Conclusion: FoxM1 was a possible EMT transcription factor regulating and controlling EMT of T24 cell line. The knockdown of FoxM1 gene expression inhibited the bladder cancer T24 cell from converting from normal epithelial phenotype cell to mesenchymal phenotype cell.