Study On The Mechanism Of ZEB1in The Regulation Of EMT And ESRP1Expression

Objective:To investigate the mechanism of ZEB1in the regulation of EMT and ESRPl expression.Methods:1. We stimulated A549cell with TGF-β(5ng/ml). Western blotting was used to detect the expression of ZEB1, E-cadherin, ESRP1, N-cadherin and fibronectin. A549cells were infected with LV-ZEB1-siRNA or LV-NC-siRNA complying with the manufacturer’s protocol. Experiments were divided into four groups LV-ZEB1-siRNA group, LV-NC-siRNA group, and these two groups stimulated with TGF-β. Western blotting was used to detect the expression of ZEB1, E-cadherin, ESRP1, N-cadherin and fibronectin. Migration and invasive capability were evaluated by in vitro cell wound-healing and invasion assay.2. To construct ZEB1gene eukaryotic expression plasmid vector, and transfect A549cells. Experiments were divided into three groups, experimental group (cells were transfected with pcDNA3.1/myc-His-ZEB1plasmids), negative control group (cells were transfected with pcDNA3.1/myc-His plasmids), and blank control group (untreated A549cells). Western blotting was used to detect the expression of ZEB1, E-cadherin, ESRP1, N-cadherin and fibronectin. Migration and invasive capability were evaluated by in vitro cell wound-healing and invasion assay. In order to study the effect of ZEB1in the regulation of ESRP1expression, we constructed luciferase reporter vector. Analysis of promoter activity was carried out transfecting the A549cells with the promoter fragments of ESRP1inserted in pGL4.17plasmid and phRL (Promega), to normalize transfection efficiency. Firefly and Renilla Luciferase (phRL) activities were determined after48hours. Luc activity was always normalized by RLuc activity.Results:1.(1) TGF-β can induce down-regulation of E-cadherin and ESRP1and up-regulation of N-cadherin and fibronectin in A549cells. Western blotting analysis showed the expression level of ZEB1protein in LV-ZEB1-siRNA group decreased compared with that in the LV-NC-siRNA group. Down-regulation of ZEB1blocks TGF-β-induced epithelial markers (E-cadherin and ESRP1) repression, but doesn’t block the up-regulation of mesenchymal markers (N-cadherin and fibronectin).(2) Wound-healing assay:TGF-β can promote cell migration compared to the negative control group (P<0.05). After TGF-β stimulation, the capacity of LV-ZEB1-siRNA group on migration was extremely lower than that of the negative control group (P<0.05).(3) Invasion assay:After TGF-P stimulation, the capacity of LV-ZEB1-siRNA group on invasion was extremely lower than that of the negative control group (P<0.05).2.(1) Western blotting analysis showed the expression level of ZEB1protein in experimental group increased compared with that in the negative control group and blank control group. Up-regulation of ZEB1can influence the expression of E-cadherin and ESRP1, but not mesenchymal markers.(2) Wound-healing assay:The capacity of experimental group on migration was extremely lower than that of the negative control group and blank control group (P<0.05).(3) Invasion assay:The capacity of experimental group on invasion was extremely lower than that of the negative control group and blank control group (P<0.05).(4) Luciferase Assays:The luciferase activity of experimental group was extremely lower than that of the negative control group (P<0.05).Conclusions:1. TGF-β can induce EMT.2. Down-regulation of ZEB1blocks TGF-β-induced epithelial markers (E-cadherin and ESRP1) repression, but doesn’t block the up-regulation of mesenchymal markers (N-cadherin and fibronectin) by induced TGF-β. Up-regulation of ZEB1can influence epithelial markers, but not mesenchymal markers. ZEB1appears to regulate only certain subsets of EMT markers.3. Down-regulation of ZEB1could lead to a significant decrease in migration and invasive capability of A549cells induced by TGF-β. Up-regulation of ZEB1can promote the migration and invasive capability of A549cells. ZEB1may play an important role in the invasion and metastasis carcinoma.4. Transfection of ZEB1in A549cells resulted in down-regulated the activity of ESRP1promoter; a similar decrease was observed after a48hours treatment of cells with TGF-β. These results suggest that ZEB1may bind to the ESRP1promoter and regulate the transcriptional activity of ESRP1.