Role Of Pokemon On CDK2 Gene Expression Regulation In HepG2 Cell

Objective:To investigation the effect of Pokemon on CDK2 gene expression regulation in HepG2 cells.Methods:1. Different promoter driving luciferase reporter construsts were developed and transfected into HepG-2 cells. Luciferse reporter assay was used to detect the effect of pokemon on CDK2 promote activity.2.ChIP was used to detect the binding ability of pokemon with the core sequence of the CDK2 promoter.3.Site directed mutagenesis was used to understand the potential cis elements for Pokemon biding within the promoter of CDK2 .4.Real-Time PCR and Westerm-Blot were utilized to detect the effect of Pokemon on the expression of CDK2 gene.Results:1. Luciferase reporter construst results suggested that cis-regulatory elements required for the core promoter activity of the CDK2 gene were mainly located in the 528bp region upstream of the ATG initiation site.Pokemon repressed CDK2 promoter activity obviously in HepG2 cells.2. Pokemon represented a direct binding ability with the promoter of CDK2.3. The GC-rich sequences at position -259 to -249bp and at the position -259 to-249 may be essential for the basal acivity of CDK2 promoter.4. Real-Time PCR results showed that pokemon may decrease the mRNA level of the CDK2 gene in HepG2 cell.5. Westerm-Blot results showed that pokemon may depress the protein expression of the CDK2 gene in HepG2 cellConclusion:1. Pokemon repressed CDK2 promoter activity by a direct binding ability with its promoter in HepG2 cells,the position -259 to-249 may be essential for the basal acivity of CDK2 promoter.2. Pokemon could inhibite the expression of CDK2 at both mRNA and protein level.