Effect of hepatitis B virus replication on the expression of inhibitor of differentiation family in hepatocytes

Abstract:

Objective：To investigate the effect of hepatitis B virus（HBV） replication on the expression of inhibitor of differentiation （Id） family in hepatocytes. Methods：The methods of qRT-PCR and Western blot were used to measure the expression of Id family in hepatoma cells with transient or stable HBV replication，and the results were further validated in normal liver cells transfected with PCH9-HBV1.1 HBV replicating plasmid and HBV transgenic mouse liver tissue cells. HepG2 cells were transfected with plasmids with overexpression of hepatitis B virus S protein（HBs），hepatitis B virus core protein（HBc），hepatitis B virus DNA polymerase（HBp），or hepatitis B virus X protein（HBx），the change in the expression of Id family was measured in these groups and the vector control group，and the regulatory effect of four HBV-en－coded proteins on Id family was analyzed and compared. Lu－ciferase reporter gene assay was used to analyze the effect of virus component proteins on the activity of Id promoter. Results：Com－pared with the control group，the group of HepG2 cells with transient transfection of HBV plasmids had significant reductions in the mRNA and protein expression of Id1 and Id3（mRNA：t=3.952 and 3.189，P=0.017 and 0.033；protein：t=10.532 and 4.155，P=0.000 and 0.014）；compared with the HepG2 control group，the HepG2.2.15 group had significantly lower mRNA and protein expression of Id1 and Id3（mRNA：t=5.553 and 7.211，P=0.005 and 0.002；protein：t=4.193 and 3.849，P=0.014 and 0.018）；compared with the HepAD38（Tet+） group，the HepAD38（Tet-） group had significantly lower protein expression of Id1 and Id3（t=3.052 and 3.712，P=0.038 and 0.021）. In addition，HBV replication significantly inhibited the mRNA and protein expression of Id1 and Id3 in LO2 cells and mouse liver tissue（mRNA：t=14.564，3.281，3.489，and 3.495，P=0.000，0.030，0.025，and 0.025；protein：t=5.651，5.336，4.948，and 5.149，P=0.005，0.006，0.008，and 0.007）. Among the HepG2 cells transfected with HBV-encoded protein plasmids，the HBc group had the greatest reductions in the mRNA expression of Id1 and Id3 compared with the vector control group（77.7% and 76.2%，F=9.945 and 37.528，t=6.481 and 10.915，P=0.000 and 0.000），and Western blot showed that the HBx group had the greatest reductions in the protein expression of Id1 and Id3（86.2% and 68.4%，F=38.225 and 7.159，t=12.550 and 5.295，P=0.000 and 0.001）. The dual-luciferase reporter gene assay showed that the HBc group had the greatest reduction in the promoter activity of Id1 and Id3（62.2% and 56.3%，F=16.530 and 5.210，t=5.442 and 3.222，P=0.000 and 0.019）. Conclusion：HBV can inhibit the expression of Id1 and Id3 in liver tissue and hepatocytes. HBc has the most significant effect in downregulating the mRNA expression of Id1 and Id3，while HBx has the most significant inhibitory effect on the protein expression of Id1 and Id3.