Regulation Effecting Study Of P107/Survivin Binding On Target Gene Expression In ECA109 Cells

Objective:To investigate whether there are the binding and interaction between p107 and Survivin in ECA109 cells,and to observe whether the binding of the two prot eins have the regulatory role on the expression of IKK beta.Methods:CMV-MCS-3flag-Survivin-SV40-Neomycin and CMV-MCS-3flag-p107-SV40-Neomycin vectors were constructed,and Western-blotting was used to detect the exp ression of related protein level in ECA109 cell.The protein binding was dete-ctedby using co-immunoprecipitation assay,and nuclear localization assay was applied to con firm the positioning of p107 and Survivin protein in cells,and the protein binding’s effect on promoter activity of IKKb was detected by dual luciferase reporter assay.Results: Western-blotting results showed that after the transfection with CMV-MCS-3flag-Survivin-SV40-Neomycin plasmid and CMV-MCS-3flag-p107-SV40-Neomycin in esophageal cancer ECA109 cells,Survivin protein and p107 protein with flag tags were detected with the 19 k D and 110 k D for the sizes,respectively.Also the sizes for endogenous survivin(16.5k D),and p107(107k D)were detected.The results of co-immunoprecipitation showed that binding of p107 and Survivin proteinsin esophageal carcinoma ECA109 cells.And the nuclear localization of the two proteins was determined.Luciferase reporter gene assay indicated that protein interaction between p107 and survivin could promote the expression of IKK via binding of its promoter.Conclusion: in esophageal carcinoma ECA109 cells,there are the binding and protein interaction of p107 and survivin,which positively regulate the expression of IKKβ.