Date Published: February 18, 2016
Publisher: Public Library of Science
Author(s): Jason Lamontagne, Joshua C. Mell, Michael J. Bouchard, Aleem Siddiqui.
Globally, a chronic hepatitis B virus (HBV) infection remains the leading cause of primary liver cancer. The mechanisms leading to the development of HBV-associated liver cancer remain incompletely understood. In part, this is because studies have been limited by the lack of effective model systems that are both readily available and mimic the cellular environment of a normal hepatocyte. Additionally, many studies have focused on single, specific factors or pathways that may be affected by HBV, without addressing cell physiology as a whole. Here, we apply RNA-seq technology to investigate transcriptome-wide, HBV-mediated changes in gene expression to identify single factors and pathways as well as networks of genes and pathways that are affected in the context of HBV replication. Importantly, these studies were conducted in an ex vivo model of cultured primary hepatocytes, allowing for the transcriptomic characterization of this model system and an investigation of early HBV-mediated effects in a biologically relevant context. We analyzed differential gene expression within the context of time-mediated gene-expression changes and show that in the context of HBV replication a number of genes and cellular pathways are altered, including those associated with metabolism, cell cycle regulation, and lipid biosynthesis. Multiple analysis pipelines, as well as qRT-PCR and an independent, replicate RNA-seq analysis, were used to identify and confirm differentially expressed genes. HBV-mediated alterations to the transcriptome that we identified likely represent early changes to hepatocytes following an HBV infection, suggesting potential targets for early therapeutic intervention. Overall, these studies have produced a valuable resource that can be used to expand our understanding of the complex network of host-virus interactions and the impact of HBV-mediated changes to normal hepatocyte physiology on viral replication.
Despite the availability of an effective vaccine, hepatitis B virus (HBV) infection remains a significant health concern with ~350 million people chronically infected worldwide . Approximately 25% of these chronically infected individuals will go on to develop HBV-associated hepatocellular carcinoma (HCC), the most common primary liver cancer, making chronic infection with HBV the leading risk factor for the development of HCC [1–3]. Globally, liver cancer is the second leading cause of cancer-related death, with nearly 750,000 deaths annually and an incidence to mortality ratio near 1 . Current treatment options for HBV-infected patients are limited to a small number of approved therapies, including reverse transcriptase inhibitors and interferon. Each of these treatments has its potential drawbacks, including side effects of treatment and the development of escape mutants, and no therapy has been developed that reaches the level of complete cure . A better understanding of HBV-mediated cellular changes in the context of viral replication is needed to expand our knowledge of virus-dependent factors and pathways, ultimately leading to the identification of novel therapeutic targets.
HBV is a non-cytopathic virus that establishes a chronic infection that can last for decades. For many chronically infected individuals, the endpoint of disease is the development of HBV-associated HCC. Because of this, many HBV-related studies attempt to identify HBV-mediated changes to the host cell that give the cell a more cancer-like phenotype. These studies, however, often do not take the time-frame of HCC development into consideration; if HBV causes dramatic tumorigenic changes immediately after infection, HCC likely would take weeks or maybe years to develop, instead of decades. In fact, HBV-mediated changes to any one factor or pathway are likely not dramatic, and may be overshadowed by cumulative changes over the course of disease progression. Our transcriptome analysis supports this hypothesis; at 48hr (24hr after infection) only 3% of DEG had a ≥ 2-fold HBV-mediated change, and at 72hr (48hr after infection) this was 10%. On the other hand, a more drastic cellular insult, such as isolation of hepatocytes and adaptation to culture may be expected to cause more dramatic cellular changes. This is supported by the observation that 57% of DEGs between 0hr and 24hr in uninfected PRHs have ≥ 2-fold change. The observation that HBV induces subtle changes to hepatocytes underscores the importance of working in a model system that closely mimics normal hepatocyte physiology. This is an important aspect of our studies, which were specifically designed as an unbiased approach to identify HBV-mediated changes to primary hepatocytes that occur early after an HBV infection and, although we are limited by the experimental system in our interpretation of long-term changes, may influence both our understanding of HBV replication and downstream HBV-associated disease. Hence, a goal of these studies was to provide a better understanding of the complex network of host-virus interactions in HBV-infected hepatocytes.