Study On Core Promoter Region And Trans-activation Of PGK1

Hepatitis B virus (HBV) is one of the major pathogens of chronic hepatitis, which often leads to serious liver disease, including cirrhosis and hepatocellular carcinoma (HCC). In the formation of HCC, Chronic hepatitis B virus infection is a major risk factor for hepatocellular carcinoma. Recent studies indicate that glycolysis is closly related to the formation of tumor and prognosis. Phosphoglycerate kinase (PGK) is the key enzyme in glycolysis. In the normal liver tissues, PGK1 protein level is significantly lower than that in cirrhosis and hepatocellular carcinoma, HCC patients with the enhanced role of glycolysis, suggesting that PGK1 expression level can be used as independent prognostic indicator for HCC patients.Purpose:This study is to confirm HBx protein on the trans-activation of PGK1 by using real-time fluorescence PCR; to inspect the influence of HBxAg on promoter activity, and to check that whether SP1 binding sites in the core promoter can bind to the nuclear protein from hepatocellular related cells.Method:We compare HepG2.2.15 cells and HepG2 cells at the mRNA level of PGK1 change using real-time fluorescence quantitative PCR technique. We constructed PGK1 fluorescent reporter vector and determined the active region by analyzing its activity, and choose gel mobility shift assay (EMSA) to verify the core promoter region of SP1 sites and the PGK1 promoter binding activity, and detect the site-directed mutagenesis changes.Results:We obtained the results in the following four respects in this study. Firstly, HepG2.2.15 cells express 3.6-fold PGK1 mRNA than than that of human hepatoma HepG2 cells. Twenty-four hours after the transfection into the HepG2 cells with pcDNA3.1 (-)-HBx, measured by a real-time fluorescence quantitative PCR, the expression of PGK1 gene mRNA levels is found 2.26-fold increase. Secondly, PGK1-promoter 1-9 series of luciferase reporter plasmids truncated body is successfully constructed, and confirmed by DNA sequencing analysis that the insertion sequence consistent with the one theoretically expressed in HepG2 cells. We successfully targeted PGK1 core promoter region. Thirdly, PGL4.10-PGK1-promoter1 with pcDNA3.1 (-)-HBx, and pcDNA3.1 (-) respectively are co-transfected in human hepatoma HepG2 cells, and the transcriptional activity of the test group increased 1.86-fold are compared to the control group. Fourthly, the study have found that SP1 sites possessing the PGK1 promoter-binding activity and loss of its activity after site-directed mutagenesis.conclusion:This study shows the following innovative aspects. HBV can up-regulate the PGK1 gene expression via HBxAg; PGK1 gene minimal promoter activity area is located at 152bp upstream of the transcription initiation site (-247bp--95bp); and Spl transcription factor involved in PGK1 transcription process.This sdudy suggest that HBx protein trans-activate the PGK1 gene, HBx enhance the transcriptional activity of PGK1 promoter and SP1 sites be involved in the transcriptional regulation of PGK1, PGK1, as the glycolytic protein in the pathogenic mechanism of HBx provide a valuable information for PGK1 as a prognosis indicators of HBV-related HCC.