The Effect And Mechanism Of Twist1 In The Development Of Endometrial Carcinoma

Objective:To explore the effect and mechanism of Twist1 in the development of endometrial carcinoma.Method:Human endometrial carcinoma Ishikawa cells(ISK)were infected with Twist1 shRNA interference fragment to construct the stable cell lines which down-regulated the Twist1 gene.1.To observe the green fluorescent protein(GFP)expression by fluorescence microscope,confirm the transfection efficiency.2.The expression of Twist1 protein in the experimental group(the ISK cells were transfected with Twist1-shRNA interference fragment),the negative control group(the ISK cells were transfected with shRNA invalid fragment)and the normal control group were determined by Western Blotting assay.3.The cell proliferation of the experimental group、the negative control group and the normal control group were detected by CCK-8 assay.4.The cell migration ability change of the experimental group,the negative control group and the normal control group were detected by wound healing assay.5.The cell migration ability and the invasion ability of the experimental group、the negative control group and the normal control group were analyzed by transwell chamber.6.The changes of cell shape of the experimental group,the negative control group and the normal control group were observed by microscope.7.The expressions of E-cadherin and VE-cadherin protein of the experimental group,the negative control group and the normal control group were determined by Western Blotting assay.8.The activation of PI3K/AKT pathway of the experimental group,the negative control group and the normal control group were determined by Western Blotting assay.Results:1.The green fluorescence was observed by fluorescence microscope in the stable ISK cell lines which down-regulated the Twist1 gene,the transfection efficiency is more than 70%.2.The experimental group compared to the negative control group and the normal control group,Twist1 expression was significantly decreased as shown by Western Blotting(P<0.05).3.By using the CCK-8 assay,the cell proliferation of the experimental group was significantly lower than the negative control group and the normal control group(P<0.05).4.The cell migration observed by wound healing assay,at 0h,the distance of experimental group,the negative control group and the normal control group were almost equal,with times going,the distance of the negative control group and the normal control group were significantly than the experimental group,the preliminary results showed that down-regulated Twist1 can inhibit the migration of ISK cells.5.Transwell chambers migration and invasion experiment results show that the perforated cell number of the experimental group was significantly less than the negative control group and the normal control group,the results showed that down-regulated Twist1 may suppress the migration and invasion ability of ISK cells.6.Compared to the negative control group and the normal control group,the surface of experimental group cells did mot see the obvious pseudopodium and the edge was slippy.7.Compared to the negative control group and the normal control group,the expression of VE-cadherin was downregulated,whereas the E-cadherin wasupregulated in the experimental group as shown by Western Blotting assay(P<0.05),indicated that down-regulated Twist1 can inhibit the epithelial-mesenchymal transition(EMT).8.Western Blotting revealed that,compared with the negative control group and the normal control group,expression of PI3 K and AKT protein significantly decreased in the experimental group(P<0.05),suggested that down-regulated Twist1 may suppress the activation of PI3K/AKT pathway.Conclusion:1.Silence Twist1 expression can decrease the proliferation of the human endometrial carcinoma Ishikawa cells(ISK),at the same time,inhibit the migration and invasion ability of ISK cells.2.PI3K/AKT signaling pathway may involve in the EMT process of endometrial carcinoma cells induced by Twist1,then Silence Twist1 expression EMT process of endometrial carcinoma cells induced by Twist1,then Silence Twist1 expression inhibit the migration and invasion ability.