Impact of HBx mutations in hepatocellular carcinoma due to chronic HBV infection

Abstract

Chronic hepatitis B virus (HBV) infection is known as a major etiology of hepatocellular carcinoma (HCC) pathogenesis. HBV X (HBx) protein was known as a multifunctional regulatory protein, which interacts directly or indirectly with a variety of targets and mediates many cellular functions, including cell cycle regulation, transcriptional transactivation, regulation of signaling pathways, apoptosis, angiogenesis, and DNA repair mechanism. This study aimed to elucidate the impact of HBx mutations on HCC development. Chronically HBV infected 96 patients were enrolled in this study: 42 chronic hepatitis B (CHB) patients, 23 liver cirrhosis (LC) patients, and 31 HCC patients. Direct sequencing of the HBV X gene and in vitro functional study of significant HBx mutations were performed. Genotype analysis revealed that all the patients had genotype C2 HBV. Direct sequencing showed HBx131, HBx130, HBx5, HBx94, and HBx38 amino acid mutations were relatively frequent in HCC patients in that order. Of various mutations, HBx130+HBx131 double mutations and HBx5+HBx130+HBx131 triple mutations were significantly high in HCC patients. Older HCC patients showed a trend to have more HBx130+HBx131 double mutations and HBx5+HBx130+HBx131 triple mutations than younger HCC patients. HBx130+HBx131 double and HBx5+HBx130+HBx131 triple mutations increased the risk for HCC by 3.75 fold (95% CI = 1.101-12.768, P = 0.003), and 5.34 fold (95% CI = 1.65-17.309, P = 0.005), respectively, when HCC patients were compared with CHB patients without cirrhosis.Functionally, there were no significant differences in HBsAg and HBeAg expression in transfected cell culture supernatants with HBV construct containing wild type, HBx5 mutant, HBx130+HBx131 double mutant, and HBx5+HBx130+HBx131 triple mutant. Also the level of HBV DNA was not different among each mutant transfected cell. However, there were significantly higher levels of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activity in cells transfected with HBx5 mutant and HBx130+HBx131 double mutant than that of wild type and HBx5+HBx130+HBx131 triple mutant. HBx5 mutation and HBx130+HBx131 double mutations appear to modulate transcriptional transactivating function of the NF-κB signaling pathway. However, HBx5+HBx130+HBx131 triple mutant did not increase NF-κB activity, there seems to be another pathway to regulate NF-κB activation. In conclusion, specific HBx mutation may promote to HCC development by activating NF-κB transcriptional activity. HBx5 mutation in genotype C2 HBV seems to be a risk factor for the development of HCC and may be a useful marker for prediction of clinical progress of patients with chronic HBV infection.