Overexpression of NR6A1 inhibits HBV replication and transcription in HepG2 cells

Abstract:

Objective：To screen for the genes in the nuclear receptor family that affect the transcriptional activities of HBV enhancers or the core promoter，and to characterize the role of the initially screened gene germ cell nuclear factor（NR6A1） on the transcription and replication of HBV. Methods：cDNA was obtained by the reverse transcription of the mRNA from HepG2 cells. Different nuclear receptor genes were amplified from the cDNA and cloned into the plasmid PCH9. Then the plasmids were co-transfected with the plasmid with Renilla luciferase reporter gene driven by the core promoter of HBV（pcore-Rluc） into the HepG2 cells. The luciferase activity was assayed to screen for the nuclear receptor genes that had an effect on the transcriptional activities of the enhancers or the core promoter. Next，the plasmid of 1.3-fold HBV genome and NR6A1 were co-transfected into HepG2 cells. The intracellular HBV replication intermediates were analyzed by Southern blot，and HBV RNA was assayed by Nothern blot. Results：Among those screened genes，NR6A1 showed a most significant influence on the HBV enhancer or the core promoter，as the Rluc activity was decreased by about 80% in the NR6A1 group compared to the control group（0.198±0.009 vs. 1.000±0.319，P=0.000）. Overexpression of NR6A1 decreased the relative luciferase activity of BCP（PCH9-1744-Rluc） group by 50% compared to that in the control group（0.504±0.023 vs. 1.000±0.099，t=6.534，P=0.0226），as well as decreasing the relative luciferase activity in the BCP+Enh Ⅱ（PCH9-1636-Rluc） group by 92% compared to that in the control group（0.089±0.002 vs. 1.000±0.042，t=31.59，P=0.001）. Both of the truncated mutants of NR6A1，NR6A1DBD or NR6A1LBD，did not significantly affect the relative luciferase activity of pcore-Rluc（P>0.05）. Conclusion：Overexpression of NR6A1 inhibits the replication of hepatitis B virus by reducing the transcription activities of the core promoter and enhancer II in HepG2 cells.