【Objective】 The purpose of this study was to construct the human phosphoenolpyruvate carboxykinase (PEPCK) promoter luciferase reporter plasmid PGL3-hPCK-luc

and investigate the effect of the coactivator peroxisome proliferator-activated receptor-gamma coactivator-1α(PGC-1α)on human PEPCK promoter activity in the presence of transcription factor hepatocyte nuclear factor 4α (HNF4α).【Method】The PEPCK promoter was cloned from normal human blood genomic DNA

and inserted into the luciferase reporter vector PGL3-basic. Then different combinations of pcDNA3.1-PGC-1α

pcDNA3.0-HNF4α

and PGL3-hPCK-luc were cotransfected into human hepatoma cells HepG2 or human hepatic cells LO2. The luciferase activity was detected after culturing cells for 48 hours.【Results】It was confirmed that the human PEPCK gene promoter was successfully inserted into the vector by gel electrophoresis and sequencing analysis. The human PEPCK gene promoter luciferase activity was increased 60-fold (P < 0.05) than the control group; When cotransfected into HepG2 cells

HNF4α could enhance the human PEPCK gene promoter luciferase activity. In HepG2 cells it was 2.69 times higher than the control group(P < 0.05)

and in LO2 cells it was 3.64-fold higher (P < 0.05); PGC-1α could significantly enhance the human HNF4α activation of PEPCK promoter. The relative luciferase activity was 2.01-fold in HepG2 cells(P < 0.05) or 2.82-fold in LO2 cells(P < 0.05) higher than the group of PGL3-hPCK-luc plus HNF4α. Compared with single transfecting group

co-transfecting pcDNA3.1-PGC-1α and pcDNA3.0-HNF4α increased the PEPCK mRNA and protein levels significantly(P < 0.05).【Conclusion】The PEPCK promoter gene reporter plasmid was successfully constructed. The transcription factor HNF4α could activate the human PEPCK promoter activity

and the coactivator PGC-1α could further strengthen the role of HNF4α.