Hepatitis C virus suppresses Hepatocyte Nuclear Factor 4 alpha, a key regulator of hepatocellular carcinoma
Introduction
Hepatitis C Virus (HCV) infection is one of the most common causes of liver disease. HCV patients often develop chronic infection, which is associated with liver inflammation, fibrosis, steatosis, cirrhosis and hepatocellular carcinoma (HCC) (Adinolfi et al., 2005, Negro and Sanyal, 2009). HCV infection presents with a disturbed lipid profile as HCV uses cellular host lipids for entry, replication, virion assembly, secretion, trafficking and escape from host immune surveillance (Popescu et al., 2014, Syed et al., 2010).
HCV possesses a single-stranded, positive sense RNA genome with a nucleotide length of 9.6 kb that encodes a polyprotein which is processed by cellular and viral proteases to yield 10 structural (core, E1, E2 and p7) and non-structural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) (Bartenschlager et al., 2013, Moradpour et al., 2007). An additional HCV protein that is synthesised by a coding sequence that overlaps HCV core protein coding sequence in the +1 reading frame, core +1, has been recently identified (Kotta-Loizou et al., 2015, Vassilaki and Mavromara, 2009). The main role for core protein in the viral life cycle is to form a nucleocapsid to protect the viral genome. Non-structural proteins are believed to comprise the viral replicase complex and mediate all the functions necessary for the replication of HCV viral genome (Lohmann et al., 1999). A considerable number of in vitro studies have suggested that expression of HCV proteins may lead to alterations of lipid metabolism and transport, cell cycle dyregulation and cellular transformation (Hoshida et al., 2014, Popescu et al., 2014). There is strong evidence that HCV proteins, including core and NS5A, are implicated in several oncogenic pathways and contribute to carcinogenesis by modulating metabolic and intracellular signalling processes (Banerjee et al., 2010). In particular, liver expression of HCV proteins in transgenic mice is linked to the development of progressive hepatic steatosis (Lerat et al., 2002, Moriya et al., 1997, Perlemuter et al., 2002) and HCC (Lerat et al., 2002, Moriya et al., 1998). Furthermore, HCV proteins directly and indirectly interact with numerous transcription factors (Banerjee et al., 2010, Tsutsumi et al., 2002) and regulate genes involved in immune phase response (Heim and Thimme, 2014, Lin et al., 2006, Polyak et al., 2001). There is sufficient evidence that HCV infection affects nuclear receptor mediated pathways and leads to problems in hepatic metabolism (Dharancy et al., 2005, Raglow et al., 2011, Wu et al., 2011). Nuclear receptors are major regulators of lipid homeostasis and inflammation; processes intricately involved in HCV infection and progression, and regulate processes essential to the progression of chronic hepatitis C.
Hepatocyte Nuclear Factor 4 alpha (HNF4α) is a member of nuclear receptor superfamily of transcription factors that is indispensable for liver development and hepatocyte differentiation and function. HNF4α is the most abundant transcription factor in the liver and a central regulator of genes involved in hepatic lipid metabolism (Hayhurst et al., 2001). HNF4α functions as master regulator of liver transcriptome by binding directly to promoters of actively transcribed genes (Odom et al., 2004). In addition to its metabolic role, HNF4α is associated with HCC. We have previously shown that HNF4α exerts a key role in hepatocellular carcinogenesis, as transient inhibition of HNF4α in immortalized hepatocytes leads to hepatocellular transformation (Hatziapostolou et al., 2011). We have also established a dynamic relationship between HNF4α and microRNAs, miR-24 and miR-629, that are direct regulators of HNF4α. Moreover, HNF4α regulates the expression of miR-124 that directly targets interleukin 6 receptor (IL6R), thus implicating HNF4α in liver inflammation and liver cancer development. Alterations in HNF4α functionality may increase the risk of development of HCC. Indeed, several mouse and human studies have shown that HNF4α expression is diminished in HCC and its loss may directly contribute to HCC development (Kalkuhl et al., 1996, Lazarevich et al., 2004, Lazarevich and Fleishman, 2008, Ning et al., 2010, Tanaka et al., 2006, Yin et al., 2008). Additionally, it was demonstrated that overexpression of HNF4α in HCC models blocks carcinogenesis and metastasis, thus indicating a potential role for HNF4α as a tumor suppressor (Ning et al., 2010, Yin et al., 2008).
In this study, we provide strong evidence that HCV remarkably decreases HNF4α expression in both protein and RNA levels. HCV proteins, core, NS5A and NS proteins, significantly suppress HNF4α expression and downregulate HNF4α promoter activity indicating regulation of HNF4α at transcriptional level. In addition, HCV leads to overexpression of miR-24 and miR-629 that we have previously shown to downregulate HNF4α expression. HCV also leads to induction of miR-124 expression and activation of IL6R-STAT3 axis. Overall, our data suggest that HCV perturbs HNF4α-microRNA pathway implying a pivotal role of HNF4α in HCV related HCC.
Section snippets
Cell lines and cell culture
For this study, Huh7 cells and the Huh7-derived cell clones, Huh7-Lunet and Huh7.25-CD81 were used. Huh7-Lunet are derivatives of Huh-7 cells cured by prolonged treatment with alpha interferon (IFN-α) of the HCV replicons and support both subgenomic and full-length HCV replication (Blight et al., 2002, Lohmann et al., 1999). Transfection experiments with viral RNA were performed in Huh7-Lunet cells, which support high level of HCV RNA replication (Friebe et al., 2005). Huh7.25-CD81 cells,
HCV infection suppresses HNF4α expression levels
HNF4α is a master transcription factor in hepatic lipid metabolism and is a major regulator of genes involved in the control of proper hepatic lipid homeostasis. Furthermore, HNF4α is a key regulator of hepatocellular carcinogenesis as its expression is diminished in HCC (Lazarevich et al., 2010, Ning et al., 2010). HNF4α interference may directly contribute to HCC development (Bonzo et al., 2012). To investigate the effect of HCV on HNF4α expression, Huh7-Lunet cells were electroporated with
Discussion
In this study, we provide strong evidence suggesting that HCV downregulates HNF4α expression at both transcriptional and translational levels. HCV remarkably reduced HNF4α protein levels in both in vitro models used (Huh7-Lunet cells electroporated with JFH1 viral genome or Huh7.25-CD81 cells infected with JC1). Moreover, HCV significantly decreased the levels of HNF4α mRNA. Overexpression of HCV proteins, core, NS5A or whole NS region, in Huh7 cells also inhibited HNF4α protein expression
Conflict of interest
The authors do not have any disclosures to report.
Author Contributions
IV: planned experiments, performed experiments, analyzed data, wrote the paper; DD: planned experiments, performed experiments; NV: performed experiments, contributed reagents or other essential materials; PM: contributed reagents or other essential materials; MHC: planned experiments, contributed reagents or other essential materials, wrote the paper.
Ackwnowledgements
We are grateful to Takaji Wakita for the JFH1 isolate and Huh7.25-CD81 cells, to Charles Rice for the 9E10 monoclonal antibody and to Ralf Bartenschlarger for Huh7-Lunet cells and subgenomic replicons expressing core protein. We would also like to thank Iannis Talianidis and Pantelis Hatzis for HNF4α-luc plasmid constructs and Athanasios Kakkanas for providing core antibody.
Financial support: This research has been co-financed by the European Union (European Social Fund-ESF) and Greek national
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