Role Of Pokemon In The Regulation Of Cdk-1 Gene Expression In Hepatocarcinoma Cells

Objective: To investigate the role of Pokemon in the regulation of CDK-1 gene expression in hepatocarcinoma cells.Methods: Cells are HepG-2 and QGY-7703 hepatocarcinoma cells. Realtime PCR was used to determine the effect of Pokemon on the transcription of CDK-1 and Western-Bloting was used to confirm the role of Pokemon in the regulation of CDK-1 expression in HepG-2 and QGY-7703 cell lines.A serial of DNA fragments at the upstream of ATG of CDK-1 gene were ligated to PGL4.10.Luciferase reporter assay was used to understand the effect of Pokemon on promoter activity of CDK--1 gene.Site directed mutation was utilized to identify the potential cis elements for Pokemon in CDK-1 promter sequence.ChIP PCR was used to comfirm the direct binding ability of Pokemon protein with core CDK-1 promote sequence .Results: In this study, we found that Pokemon expression could down regulate both the mRNA and protein level of CDK-1 in HepG-2 and QGY-7703 cells.All the pLuc-1018,pLuc-541and pLuc-242 were shown to have promoter activity,and Luciferase reporter assay showed that Pokemon could obviously down regulate the promoter activity of pLuc-1018(P<0.01),whereas ,no obvious effects of Pokemon on the activity of pLuc-541and pLuc-242 were observed in both HepG-2 and QGY-7703 cells(P>0.05). ChIP PCR results showed that Pokemon could bind with the core sequence of CDK-1 promoter directly in both HepG-2 and QGY-7703 cells.Conclusion: Pokemon down regulates the expression of CDK-1 via direct binding with the promoter and down regulating the promoter activity in both HepG-2 and QGY-7703 cells.