TGFβ1-Smad Signaling Pathway In TGFβ1 And Smad4 Effect On Bile Duct Epithelial-mesenchymal Transition Between (EMT) Of

ObjectiveTo investigate the effects of TGFβ1and Smad4in TGF β1-Smad signal transduction pathway on the expression of Snail gene, Slug gene, Vimentin and E-Cadherin, so as to define the role of TGFβ1and Smad4gene in the process of epithelial mesenchymal transition (EMT) in cholangiocarcinoma and provid a theoretical basis for the drug therapy of cholangiocarcinoma.MethodFirst, Pre-experiment:Firstly, using the Western Blot method to verify the expression of the Smad4, E-Cadherin, Vimentin in cholangiocarcinoma cell line RBE. Secondly, to test the infection efficiency of plvt7lentiviral for cholangiocarcinoma cell line RBE, and screen the minimum amount of virus which can made the infection efficiency reach80%at least as the experimental condition of next step. Second, Construction of Smad4gene RNAi lentiviral vector:The specific shRNA fragment were designed then synthesized according to Smad4gene, then connected to pMagic7.1plasmid, transformed E. coli. The plasmid were identified with agarose gel electrophoresis and gene sequencing. Finally lentiviral RNAi vector were packaged into HEK-293T cells. In the meantime negative control lentiviral vectors were constructed. And, making them infect the cholangiocarcinoma cell line RBE, detecting the effect of interference, to determine the next step experiment. Third, Experimental packet and test of target genes and protein:In the experimental the group were divided into6groups:RBE cells group, RNA interference cell group, NC group, RBE cells+TGFβ1treated group, RNA interference+TGFβ1treated group, NC+TGFβ1treated group. Using Real time PCR and Western Blot to detect the differences of the expression of Smad4, Snail, Slug, E-Cadherin and Vimentin in every group when cells were being cultured for24h and48h.ResultFirst, the expression of Snail and Slug gene was up-regulated and the expression of E-Cadherin and Vimentin was down-regulated after the down-regulation of Smad4gene expression in human holangiocarcinoma cell line RBE(p<0.05). Second, the expression of Vimentin, Smad4and Slug gene was up-regulated and the expression of E-Cadherin was down-regulated(p<0.05) but no significant difference between the expression of Snail gene(p>0.05) in human holangiocarcinoma cell line RBE after the concentration of was increased in cell culture fluid. Third, Smad4gene was downregulated while increasing TGFβ1concentration in the cell culture medium than Snail and Slug gene was up-regulated and the expression of E-Cadherin was down-regulated and Vimentin was down-regulated when them were at the treatment of24h and it was up-regulated when them were at the treatment of24h(p<0.05).ConclusionTGFβ1play an important role in the process of EMT in cholangiocarcinoma through TGFP signal transduction pathway, and the participation of Smad4cannot alone but required with other signaling pathway induced the EMT in cholangiocarcinoma. So that to illustrate that can interfere the mechanism of EMT in cholangiocarcinoma through regulation of TGF β1and Smad4.