Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths worldwide and is highly correlated with hepatitis virus infection. Our previous report shows that a DEAD box RNA helicase, DDX3, is targeted and regulated by hepatitis C virus (HCV) core protein, which implicates the involvement of DDX3 in HCV-related HCC development. In this study, the potential role of DDX3 in hepatocarcinogenesis is investigated by examining its expression in surgically excised human HCC specimens. Here we report the differential deregulation of DDX3 expression in hepatitis virus-associated HCC. A significant downregulation of DDX3 expression is found in HCCs from hepatitis B virus (HBV)-positive patients, but not from HCV-positive ones, compared to the corresponding nontumor tissues. The expression of DDX3 is differentially regulated by the gender and, moreover, there is a tendency that the downregulation of DDX3 expression in HCCs is more frequent in males than in females. Genetic knockdown of DDX3 with small interfering RNAs (siRNA) in a nontransformed mouse fibroblast cell line, NIH-3T3, results in a premature entry to S phase and an enhancement of cell growth. This enhanced cell cycle progression is linked to the upregulation of cyclin D1 and the downregulation of p21 WAF1 in the DDX3 knockdown cells. In addition, constitutive reduction of DDX3 expression increases the resistance of NIH-3T3 cells to serum depletion-induced apoptosis and enhances the ras-induced anchorage-independent growth, indicating the involvement of DDX3 in cell growth control. These findings together with the previous study suggest that the deregulation of DDX3, a DEAD box RNA helicase with cell growth-regulatory functions, is involved in HBV-and HCV-associated pathogenesis.

Two retroviral vectors carrying an antisense gene from the hepatitis B virus (HBV) preS/S or preC/C were constructed and used to infect the human hepatoblastoma cell line 2.2.15, which expresses HBV surface antigen (HBsAg), HBV e antigen (HBeAg) and releases HBV particles. The results showed that the inhibitory effects of antisense gene transfer, mediated by retroviral vectors on the expression of HBV antigens, appeared as early as day 3 after transduction, reached a maximum on day 5 and persisted for at least 11 days. Our data indicate that, on day 5 after introduction, antisense preS/S inhibited HBsAg and HBeAg expression by 71% and 23%, and the antisense preC/C inhibited HBsAg and HBeAg expression by 23% and 59%. HBV DNA production, in the supernatant of the 2.2.15 cells transduced with either antisense preS/S or preC/C, was also reduced on day 5, but the viability of the 2.2.15 cells was not affected. Our results demonstrate that the replication and expression of HBV can be inhibited through antisense gene transfer mediated by retroviral vectors and that the antisense-preC/C or antisense-preS/S may be potentially useful for clinical gene therapy against HBV.