Hint1 over expression inhibits the activity of AP-1 transcription factor in HepaG2 cells / 中华普通外科杂志

ABSTRACT

Objective To study the inhibition of AP-1 transcription factor activity by Hint1 gene over expression in HepG2 cell lines. Methods The Hintl gene was amplified, and then was inserted into the pcDNA3/HA eukaryotic expression plasmid. The constructed pHA-Hint1 plasmid was confirmed by DNA sequencing. The pHA-Hint1 was transfected into the HepG2 human hepatoma cells. Semi-quantitative RT-PCR and Western-blot were used to detecte the expression of HA-Hint1. The HepG2 cells were co-transfected with pHA-Hint1 and pAP-1/Luc luciferase reporter. At 36 h after transfection, luciferase assay system was used to detect the AP-1 transcription factor activity. Results The constructed pHA-Hint1 was confirmed by DNA sequencing, pHA-Hint1 gene transduction through lipofectine induced over-expression in HA-Hint1 mRNA (t =3.89, P<0.05) and HA-Hintl protein (t=3. 12, P<0.05). Co-transfection of Hint1 gene inhibits AP-1 luciferase activity. Cotransfection with increased concentration of a pHA-Hint1 plasmid (0 μg/ml, 0. 5 μg/ml, 1.0 μg/ml, 1.5 μg/ml, 2. 0 μg/ml) produced a concentration-dependent inhibition of AP-1 transcription factor activity. At the concentration of 1.5 μg/ml, and 2.0 μg/ml, the activity inhibition reaches significant difference ( F = 72. 009, P < 0. 05 ). Conclusion Over-expression of Hintl can, at least in part, inhibit the AP-1 transcription factor activity in HepG2 cells.