Background & aims: The human hepatitis B virus (HBV) is a major cause of chronic hepatitis and hepatocellular carcinoma, but molecular mechanisms driving liver disease and carcinogenesis are largely unknown. We therefore studied cellular pathways altered by HBV infection.
Methods: We performed gene expression profiling of primary human hepatocytes infected with HBV and proved the results in HBV-replicating cell lines and human liver tissue using real-time polymerase chain reaction and Western blotting. Activation of signal transducer and activator of transcription (STAT3) was examined in HBV-replicating human hepatocytes, HBV-replicating mice, and liver tissue from HBV-infected individuals using Western blotting, STAT3-luciferase reporter assay, and immunohistochemistry. The consequences of STAT3 activation on HBV infection and cell survival were studied by chemical inhibition of STAT3 phosphorylation and small interfering RNA-mediated knockdown of STAT3.
Results: Gene expression profiling of HBV-infected primary human hepatocytes detected no interferon response, while genes encoding for acute phase and antiapoptotic proteins were up-regulated. This gene regulation was confirmed in liver tissue samples of patients with chronic HBV infection and in HBV-related hepatocellular carcinoma. Pathway analysis revealed activation of STAT3 to be the major regulator. Interleukin-6-dependent and -independent activation of STAT3 was detected in HBV-replicating hepatocytes in cell culture and in vivo. Prevention of STAT3 activation by inhibition of Janus tyrosine kinases as well as small interfering RNA-mediated knockdown of STAT3-induced apoptosis and reduced HBV replication and gene expression.
Conclusions: HBV activates STAT3 signaling in hepatocytes to foster its own replication but also to prevent apoptosis of infected cells. This very likely supports HBV-related carcinogenesis.
Keywords: APR, acute phase response; Apoptosis; CRP, C-reactive protein; DMSO, dimethyl sulfoxide; FCS, fetal calf serum; HBV pg RNA, hepatitis B pregenomic RNA; HBV, Hepatitis B virus; HBVtg, hepatitis B transgenic; HBeAg, hepatitis B early antigen; HCC, hepatocellular carcinoma; HNF, hepatocyte nuclear factor; Hepatitis B Virus Infection; Hepatocellular Carcinoma; IFN, interferon; IL-6, interleukin 6; IRF3, interferon regulatory factor 3; NAC, N-acetyl-L-cysteine; PCR, polymerase chain reaction; PHH, primary human hepatocyte; ROS, reactive oxygen species; RT, reverse transcription; STAT3 Signaling; STAT3, signal transducer and activator of transcription 3; cDNA, complementary DNA; cRNA, complementary RNA; cccDNA, covalently closed circular DNA; mRNA, messenger RNA; p.i., postinfection; pSTAT3, phosphorylated signal transducer and activator of transcription 3; pgRNA, pregenomic RNA; siRNA, small interfering RNA.