The Role Of Notch-1for Epithelial-mesenchymal Transition In Esophageal Squamous Cell Carcinoma

Objective:TGF-β1induced esophageal squamous cancer cells (Eca-109) that occurred EMTby DAPT down-regulating Notch-1signaling pathway to explore the role of Notch-1for epithelial-mesenchymal transition in esophageal squamous cell carcinomas.Methods:1.TGF-β1induced esophageal squamous cell carcinomas (Eca-109) that occurredEMT by adding0ng/ml,0.5ng/ml,1ng/ml,2ng/ml,5ng/ml,10ng/ml TGF-β1andscreen ing optimal induction concentration of TGF-β1. Cell viability was detectedafter48h by CCK-8, the expression change of E-cadherin and Vimentin weredetected by Western-blotting.2.To explore the effect of Notch-1signaling pathway in Eca-109-EMT byspotting0μM,5μM,10μM,15μM,20μM DAPT, and screening the best inhibitoryconcentrations of DAPT, Cell viability was detected after48hours by CCK-8, theexpression change of E-cadherin and Vimentin were detected by Western-blotting.3.To explore the role of Notch-1for epithelial-mesenchymal transition inesophageal squamous cell carcinomas by DAPT down-regulating Notch-1signalingpathway.The test was divided into Control, DMSO, DMSO+TGF-β1, TGF-β1,TGF-β1+DAPT, DAPT. The expression of E-cadherin and Vimentin were detectedafter48hours by the IF, and the expression change of Notch-1ICDand Hes-1weredetected by Western-blotting;the clone were dyed by crystal violet after4W, and thenit were counted; the migration of Eca-109were observed in0hour,6hours,12hours,24hours.Results:1.The Western-blotting test showed that expression of E-Cadherin wassignificantly reduction in TGF-β1-5ng/ml group, compared with Control,0.5,1,2ng/ml group (P<0.05), compared with10ng/ml group, it was no significant difference(P>0.05); the expression of Vimentin was significantly increment in TGF-β1-5ng/ml group, Compared with Control group, compared with Control,0.5,1,2ng/ml group(P<0.05), compared with10ng/ml group, it was no significant difference (P>0.05).The best induced concentration of Eca-109-EMT was5ng/ml by TGF-β1。2.The expression of E-Cadherin was significantly increment in DAPT(15μmol/ml)+TGF-β1(5ng/ml)group,compared with Control, DAPT(5/10μmol/ml)+TGF-β1(5ng/ml)group(P<0.05), compared with DAPT(20μmol/ml)+TGF-β1(5ng/ml),it was no significant difference(P>0.05); the expression of Vimentin was significantlyreduction in DAPT(15μmol/ml)+TGF-β1(5ng/ml)group, DAPT(5/10μmol/ml)+TGF-β1(5ng/ml)group(P<0.05), compared with DAPT(20μmol/ml)+TGF-β1(5ng/ml), itwas no significant difference(P>0.05). The best inhibited concentration of Eca-109-EMT was15μmol/ml by DAPT.3.The expression of Notch-1ICDand Hes-1was significantly increment in TGF-β1-5ng/ml group, compared with DMSO(5μl)+TGF-β1(5ng/ml)(P>0.05), comparedwith Control (0μmol/ml), DMSO(5μl), DAPT(15μmol/ml)+TGF-β1(5ng/ml),DAPT(15μmol/ml), that was a significant difference(P<0.05); The expression ofNotch-1ICDand Hes-1was significantly reduction in DAPT(15μmol/ml)+TGF-β1(5ng/ml),compared with Control (0μmol/ml), DMSO(5μl), DMSO(5μl)+TGF-β1(5ng/ml), TGF-β1(5ng/ml), DAPT (15μmol/ml), that was a significantdifference (P<0.05).4.The cell immunofluorescence results suggest that the expression of E-cadherinwas in the cell membrane, Vimentin was in the cytoplasm, the nucleus was also asmall amount of expression.5.The Cell clone formation rate of test results showed that the clone formationrate and the number of Eca-109was significantly increment in TGF-β1-5ng/ml group,compared with DMSO(5μl)+TGF-β1(5ng/ml)(P>0.05), compared with Control(0μmol/ml), DMSO(5μl), DAPT(15μmol/ml)+TGF-β1(5ng/ml), DAPT(15μmol/ml),that was a significant difference (P<0.05); The clone formation rate and the numberof Eca-109was significantly reduction in DAPT(15μmol/ml)+TGF-β1(5ng/ml),compared with Control (0μmol/ml), DMSO(5μl), DMSO(5μl)+TGF-β1(5ng/ml), TGF-β1(5ng/ml), DAPT (15μmol/ml), that was a significant difference (P<0.05).6.The migration Assay showed that the migration distance of Eca-109was significantly increment in TGF-β1-5ng/ml group, compared with DMSO(5μl)+TGF-β1(5ng/ml)(P>0.05), compared with Control(0μmol/ml), DMSO(5μl), DAPT(15μmol/ml)+TGF-β1(5ng/ml), DAPT(15μmol/ml), that was a significant difference(P<0.05); The migration distance of Eca-109was significantly reduction in DAPT(15μmol/ml)+TGF-β1(5ng/ml), compared with Control (0μmol/ml), DMSO(5μl),DMSO(5μl)+TGF-β1(5ng/ml), TGF-β1(5ng/ml), DAPT(15μmol/ml), that was asignifycant difference (P<0.05).Conclusion:The esophageal squamous cancer cells-EMT of TGF-β1inducement wasinhibited by DAPT down-regulating Notch-1signaling pathway, and the proliferationand migration of cell were also reduced.