Gene expression analysis in HBV transgenic mouse liver: a model to study early events related to hepatocarcinogenesis

Abstract

Hepatitis B virus (HBV) is one of the major etiological factors responsible for the development of hepatocellular carcinoma (HCC). We used a transgenic mouse, containing HBV sequences, as a model system to unravel the molecular mechanisms of hepatocarcinogenesis induced by HBV. We chose this animal model because it consistently develops liver cancer after intermediate steps that mimic the natural history of HBV infection in humans. In this study, we focus our attention on the early events leading to liver cancer. We compared the gene expression profile of 3-month-old transgenic mice with that of 3-month-old wild-type (wt) animals. In the transgenic mouse, microarray data analysis showed a total of 45 significantly differentially expressed genes, 25 highly expressed (fold change > or =2; P = 0.0025), and 20 downregulated (fold change < or =0.5; P = 0.0025). These genes belong to several different functional categories such as the regulation of immunological response, transcription, intracellular calcium ion mobilization, regulation of cell cycle and proliferation, NF-kappab signal transduction cascades, and apoptosis. In particular, the upregulation of the antiapoptotic gene NuprI and the downregulation of the proapoptotic gene Bnip3 were found. This observation was supported by an in vitro apoptosis assay that showed downregulation of apoptosis in hepatocytes of HBV transgenic mouse compared with wt mice treated with staurosporine. In conclusion, our experimental approach allowed identification of new genes modulated by HBV and showed that the apoptotic process was deregulated in transgenic mouse hepatocytes. These data shed light on one possible mechanism by which HBV induces hepatocarcinogenesis.

Figure 1

Semiquantitative RT-PCR analysis. Semiquantitative RT-PCR analysis was performed on total RNAs prepared from livers of 3-month-old wt (control) and HBV transgenic mice. Nupr1 (nuclear protein 1), Cdkn2d (p19), and Car2 (carbonic anhydrase) expression levels were analyzed. β-Actin mRNA amplification was performed as an internal control. The reaction without cDNAs was the negative control. The results were concordant to microarray data.

Figure 2

Hmgb2 protein expression in wt and HBV transgenic mice livers. Total protein extracts (50 μg of total protein/lane) were prepared from livers isolated from wt (control) and HBV transgenic mice at 3 months of age. All samples were analyzed by SDS-PAGE and immunoblotting with anti-Hmgb2 and anti–β-tubulin antibodies.

Figure 3

Apoptotic assay. The DNA-laddering assay was performed on primary cultured hepatocytes isolated from 3-month-old wt (lanes 2–5) and HBV transgenic (lanes 6–9) mice in the presence or absence of 10 μM staurosporine.