Effect Of Transforming Growth Factor-smad Signaling Pathway On Epithelial-mesenchymal Transition In RBE Cells

Objective: To investigate the role of transforming growth factor β-Smad related signaling pathways on epithelial mesenchymal transition of cholangiocarcinoma cells.Methods:Four or six subgroups were set up for the treatment of cholangiocarcinoma RBE cells according to single or combination therapy(TGFβ1,SB431542,SB525334).(1)Observe the morphological changes of RBE cells in each group.(2)The Trasswell assay was used to detect the migration and invasiveness of RBE cells.(3)The protein expression of Smad4,E-cadherin,N-cadherin and Slug were detected by Western blot.Results:1.Compared with the blank control group,the cell morphology of TGFβ1group changed significantly.The cells were elongated and spindle-shaped,showing an EMT-like trend.However,the morphology of cells in SB525334 group and SB431542 group had no significant changes compared with the control group.2.RBE cell migration and invasion assays.(1)Migration assays: The number of RBE cells that passed the Transwell cells was(85.66±7.76)in the Control group,(118.17±12.69)in the TGFβ1group,(53.00±5.51)in the SB431542 group,(86.33±9.67)in the TGFβ1+SB431542 group,and(52.50±7.45)in the SB525334,TGFβ1+SB525334(81.00±7.62).Compared with the Control group,the number of cells in the TGFβ1 group was significantly increased,and the difference was statistically significant(*P<0.05).Compared with the Control group,the number of cells in the SB525334/SB431542 group was significantly decreased,and the difference was statistically significant(*P<0.05)(2)Invasive assays: The number of RBE cells that passed the Transwell cells was(73.83±9.47)in the control group,(127.83±7.33)in the TGFβ1group,(59.83±7.28)in the SB431542 group,(79.67±7.31)in the TGFβ1+SB431542 group,and(55.67±7.26)in the SB525334 group,TGFβ1+SB525334(76.00±8.51).Compared with the Control group,the number of cells in the TGFβ1 group was significantly increased,and the difference was statistically significant(*P<0.05).Compared with the Control group,the number of cells in the SB525334/SB431542 group was significantly decreased,and the difference was statistically significant(*P<0.05).3.Smad4 protein and E-cadherin,N-cadherin,Slug protein expression.(1)Changes of Smad4 protein expression: Compared with the Control group,the expression level of Smad4 protein was significantly higher in the TGFβ1 group,and Smad4 protein showed a decreasing trend in the SB525334/SB431542 group.(2)Changes of E-cadherin,N-cadherin,and Slug protein expression:Compared with the control group,E-cadherin protein expression was significantly decreased in the TGFβ1 treated group,and N-cadherin and Slug protein were significantly increased in the mesenchymal marker.In the SB431542/SB525334 group,the E-cadherin protein showed an increasing trend compared with the control group,while the N-cadherin and Slug proteins decreased significantly.Conclusions:1.TGFβ1 can induce morphological EMT transformation in RBE cells.2.TGFβ1 can significantly increase the migration and invasion of REB cells,while SB525334/SB4315442 can block this effect.3.TGFβ1 can promote the expression of Smad4 protein,promote the expression of N-cadherin and Slug protein,and inhibit the expression of E-cadherin protein.TβRⅠ inhibitor(SB431542/SB525334)can promote the expression of E-cadherin protein,inhibit the expression of N-cadherin and Slug protein and exert the effect of inhibiting EMT.