Globally, hepatocellular carcinoma (HCC) is the sixth most common cancer. A lack of early detection methods and effective treatment options also make it one of the most deadly. Despite its prevalence, much remains unknown about the molecular mechanisms involved in the development of HCC. It is known however, that chronic infection with the hepatitis B virus (HBV) is one of the leading risk factors for developing HCC. Three main factors are proposed to play a role in the development of HBV-associated HCC: chronic inflammation resulting in hepatocyte destruction and regeneration;deleterious effects of HBV DNA integration into the host genome;and the impact of expression of HBV proteins, specifically the hepatitis B virus X protein (HBx). HBx is a multifunctional non-structural protein that studies have shown can have either direct or cooperative oncogenic potential. HBx can also activate transcription factors such as NF-kB. Normal activation of NF-kB causes an immediate-early response resulting in strong activation of pro-survival and inflammatory factors. NF-kB also is involved in the regulation of expression of multiple microRNAs (miRNAs), which are small ~22nt RNAs that can act as post-transcriptional regulators of mRNAs. Both NF-B and miRNAs have been implicated in cancer, and studies have successfully utilized alterations in the miRNA profile as biomarkers of certain diseases such as cancers and viral infections. The proposed studies will examine the role of NF-kB in the HBx-mediated alteration of the hepatocyte miRNA profile, and how these changes influence the development of HCC. Particular attention will be paid to a specific panel of miRNAs based on their association with HBV, HBx, or HBV-associated HCC in addition to their critical role in regulating cellular processes such as proliferation and the immune response.
In Aim 1, HBx- induced changes in miRNA expression levels will be determined by miRNA microarray and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) studies. NF-kB-related changes to this expression pattern will then be determined by inhibiting NF-B activation with a NF-kB super-repressor. Importantly, these studies will be done in freshly isolated, cultured primary rat hepatocytes, which are a more likely to retain their physiological miRNA profile than immortalized cell lines.
I Aim 2, a transcriptome-wide analysis of miRNA-bound mRNAs will allow us to identify a profile of all mRNAs actively being regulated by miRNAs, which will be validated by subsequent target mRNA and protein analysis.
In Aim 3, progressive changes to miRNA expression and proposed mRNA targets will be confirmed in vivo over the course of HCC development in an HBx-transgenic mouse model that develops HCC. Manipulation of extensively regulated mRNA targets will confirm their role, and the role of altered miRNA regulation, in the development of HCC. Together, these studies will support the hypothesis that NF-kB influences the HBx-mediated alteration of the miRNA profile in hepatocytes, and that these changes directly alter regulation of important cellular targets involved in the development of HCC. These studies will aid in the long-term goal of determining molecular mechanisms involved in the development of HCC, and potentially identify novel therapeutic targets and diagnostic markers for preventing or detecting HBV-associated HCC.

Public Health Relevance

Inflammation plays a significant role in development of hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC). The HBV X protein (HBx) activates NF-kB, the main mediator of inflammation, and both NF- kB activation and HBx expression have been implicated in HCC development. We will investigate the role of NF-kB in HBx-mediated changes in hepatocyte miRNA levels and the influence of these changes on the development of HBV-associated HCC.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1-F09-A (08))
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Schmidt, Michael K
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Drexel University
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Lamontagne, R Jason; Casciano, Jessica C; Bouchard, Michael J (2018) A broad investigation of the HBV-mediated changes to primary hepatocyte physiology reveals HBV significantly alters metabolic pathways. Metabolism 83:50-59
Mehta, Anand; Comunale, Mary Ann; Rawat, Siddhartha et al. (2016) Intrinsic hepatocyte dedifferentiation is accompanied by upregulation of mesenchymal markers, protein sialylation and core alpha 1,6 linked fucosylation. Sci Rep 6:27965
Lamontagne, Jason; Mell, Joshua C; Bouchard, Michael J (2016) Transcriptome-Wide Analysis of Hepatitis B Virus-Mediated Changes to Normal Hepatocyte Gene Expression. PLoS Pathog 12:e1005438
Lamontagne, R Jason; Bagga, Sumedha; Bouchard, Michael J (2016) Hepatitis B virus molecular biology and pathogenesis. Hepatoma Res 2:163-186
Lamontagne, Jason; Steel, Laura F; Bouchard, Michael J (2015) Hepatitis B virus and microRNAs: Complex interactions affecting hepatitis B virus replication and hepatitis B virus-associated diseases. World J Gastroenterol 21:7375-99