The maintenance of terminally differentiated cells, especially hepatocytes, in vitro has proven challenging. Here we demonstrated the long-term in vitro maintenance of primary human hepatocytes (PHHs) by modulating cell signaling pathways with a combination of five chemicals (5C). 5C-cultured PHHs showed global gene expression profiles and hepatocyte-specific functions resembling those of freshly isolated counterparts. Furthermore, these cells efficiently recapitulated the entire course of hepatitis B virus (HBV) infection over 4 weeks with the production of infectious viral particles and formation of HBV covalently closed circular DNA. Our study demonstrates that, with a chemical approach, functional maintenance of PHHs supports long-term HBV infection in vitro, providing an efficient platform for investigating HBV cell biology and antiviral drug screening.

The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as the hepatitis B virus (HBV) receptor enabled researchers to create hepatoma cell lines susceptible to HBV infection. Infection in current systems, however, is inefficient and virus fails to spread. Infection efficiency is enhanced by treating cells with polyethylene glycol 8000 (PEG) during infection. However, this alone does not promote virus spread. Here we show that maintaining PEG in culture medium increases the rate of infection by at least one order of magnitude, and, most importantly, promotes virus spread. To demonstrate the utility of this system, we show that two interferon-stimulated genes (ISGs), ISG20 and tetherin, restrict HBV spread in NTCP-expressing hepatoma cells. Thus, this protocol can be easily applied to existing cell culture systems to study the complete HBV life cycle, including virus spread.

Long noncoding RNAs (lncRNA) are being implicated in the development of many cancers. Here, we report the discovery of a critical role for the lncRNA GCASPC in determining the progression of gallbladder cancer. Differentially expressed lncRNAs and mRNAs between gallbladder cancer specimens and paired adjacent nontumor tissues from five patients were identified and validated by an expression microarray analysis. Quantitative real-time PCR was used to measure GCASPC levels in tissues from 42 gallbladder cancer patients, and levels of GCASPC were confirmed further in a separate cohort of 89 gallbladder cancer patients. GCASPC was overexpressed or silenced in several gallbladder cancer cell lines where molecular and biological analyses were performed. GCASPC levels were significantly lower in gallbladder cancer than adjacent nontumor tissues and were associated with tumor size, American Joint Committee on Cancer tumor stage, and patient outcomes. GCASPC overexpression suppressed cell proliferation in vitro and in vivo, whereas GCASPC silencing had opposite effects. By RNA pull-down and mass spectrometry, we identified pyruvate carboxylase as an RNAbinding protein that associated with GCASPC. Because GCASPC is a target of miR-17-3p, we confirmed that both miR-17-3p and GCASPC downregulated pyruvate carboxylase level and activity by limiting protein stability. Taken together, our results defined a novel mechanism of lncRNA-regulated cell proliferation in gallbladder cancer, illuminating a new basis for understanding its pathogenicity. Cancer Res; 76(18); 5361-71. Ó2016 AACR.

Background & Aims As important virological markers, serum HBsAg and HBV DNA levels show large fluctuations among chronic hepatitis B (CHB) patients. The aim of this study was to reveal the potential impact and mechanisms of amino acid (AA) substitutions in small hepatitis B surface proteins (SHBs) on serum HBsAg and HBV DNA levels. Methods Serum samples from 230 untreated chronic hepatitis B patients with genotype C HBV were analyzed in terms of HBV DNA levels, serological markers of HBV infection and SHBs sequences. In vitro functional analysis of the identified SHBs mutants was performed. Results Among 230 SHBs sequences, there were 39 (16.96%) sequences with no mutation detected (wild-type, WT) and 191 (83.04%) with single or multiple mutations. SHBs consist of 226 AAs, of which 104 (46.02%) had mutations in our study. Some mutations (e.g. sE2G, sL21R, sG24K, sT47A/K, sC69stop (sC69*), sL95W, sL98V, and sG145R) negatively correlated with serum HBsAg levels. HBsAg and HBV DNA levels from this group of patients had a positive correlation (r = 0.61, p < 0.001). In vitro analysis showed that these mutations reduced extracellular HBsAg and HBV DNA levels by restricting virion secretion and antibody binding capacity. Virion secretion could be rescued for sE2G, sC69*, and sG145R by co-expression of WT HBsAg. Conclusion The serum HBsAg levels were lower in untreated CHB patients with novel SHBs mutations outside the major antigenic region than those without mutations. Underlying mechanisms include impairment of virion secretion and lower binding affinity to antibodies used for HBsAg measurements.