Department of Pathology

EXPERIMENTAL PATHOLOGY

Our goal of our research program is to understand the role of microRNAs and DNA methylation machinery in hepatocellular cancer (HCC), the most prevalent liver cancer. Our research focuses on gaining mechanistic insight on the molecular basis of HCC that could identify novel biomarkers for diagnosis/prognosis of the disease, and developing agents for prevention and therapy of liver cancer in pre-clinical models. Our study could be translated to human HCC patients in collaboration with oncologists at The Ohio State University Comprehensive Cancer Center. The ongoing projects in the lab are the following.

1. Elucidating the role of miR-122 in liver homeostasis using primary HCCs of human and rodent origins aw well as novel mouse models
MicroRNAs (miRs) are small noncoding RNAs that function in negatively regulating gene expression of their targets post-transcriptionally. miR-122 is a liver-specific miRNA that is most abundantly expressed miRNA in humans. We were first to report decrease in miR-122 in primary HCCs developed in humans and rats. Subsequently, many investigators have shown that down regulation of miR-122 correlates with poor prognosis and metastasis in HCC patients. One of our goals is to elucidate the biological functions of this liver-specific tiny RNA, which is most abundant in human. To this end, we have generated conditional knockout (KO) mice using Cre-Lox system as well as liver-specific and germline KO mice (Figure 1). Using these mouse models, we confirmed that miR-122 is a bona fide tumor suppressor. It is amazing to discover that this tiny, 22 nucleotide long RNA exerts multifarious functions in the liver such as regulation of cholesterol, triglyceride and iron homeostasis, and hepatitis C and B virus replication.

Since microRNAs, in general, exert their biological functions by suppressing expression of target mRNAs we have recently identified miR-122 targets in mouse and human livers using unbiased HITS-CLIP (high throughput sequencing coupled with cross-linking immunoprecipitation) analysis in collaboration with Dr. Robert Darnell’s group at Rockefeller University. This analysis identified both canonical and non-canonical miR-122 targets in mouse and human livers. Currently, we are analyzing signaling pathways and cellular processes regulated by these targets in the liver. Functional analysis of these targets will be the key for elucidating biological function of this liver-specific microRNA and the consequence of its loss of function in promoting HCC.

We believe miR-122 knockout mouse is an ideal preclinical model to study mechanism of various liver diseases and to test novel therapeutics (Figure 2). Using liver-specific KO mouse model and human hepatocytes, we are also elucidating its role in liver injury in response to xenobiotics such as acetaminophen and carbon tetrachloride. This mouse model was instrumental in addressing several important biological questions such as the role of miR-122 in liver ploidy, siRNA hepatotoxicity, hepatotrophic virus infection and ischemia-reperfusion injury.

2. Therapeutic delivery of microRNA using nanoparticles or viral vectors
Our group also successfully developed and tested several liposomal nanoparticles for delivery of microRNA mimics or anti-miRNAs to the liver tumors in mouse models in collaboration with nanotechnology experts at OSU (Drs. L. James Lee and Robert Lee).

In addition, we have also successfully delivered miR-122 precursor gene using an AAV8 vector in an oncogene (c-MYC)-induced mouse model of HCC that dramatically inhibited tumor growth in collaboration with Dr. Joshua Mendell at UTSouthwestern(Figure 4).

Apart from miR-122, we are using different animal models to identify early molecular changes during hepatocarcingenesis in response to chemical carcinogens and tumor-promoting diets, such as choline deficient and amino acid defined (CDAA) diet and to test new therapeutics before their clinical trials in human patients. For this purpose, we are using several mouse models, including miR-155KO, miR-155 conditional knockin (cKI) mice. These animals are fed with diets that promote NAFLD (nonalcoholic fatty liver disease) and NASH (nonalcoholic steatohepatitis), a complication of metabolic syndrome in humans that often leads to liver cancer or Lieber-DeCarli’s diet that causes alcoholic liver disease. In these models, we are testing different chemo and dietary preventive agents to block initiation and progression of HCC. In parallel, we are using human primary liver tumors (from NCI’s Cooperative Human Tissue Network and OSUCCC) to validate the results obtained in mouse models to humans and vice versa.

3. Role of DNA methylation and hydroxymethylation in hepatocarcinogenesis
Another research interest of our lab is to elucidate the role of DNA methylation and hydroxymethylation in hepatocarcinogenesis. The predominant epigenetic modification, 5-methylcytosine (5-mC), essential for mammalian development, is deregulated in almost all cancers including HCC. Recently, several new epigenetic modifications, such as 5-hydroxymethyl-C (5-hmC), 5-formyl-C (5-fC) and 5 carboxy-C (5CaC) obtained from enzymatic processing of 5-mC, are identified in mammalian DNA. Sequential conversion of 5-mC to 5-caC that gets decarboxylated to cytosine is a mechanism for active DNA demethylation. However, These newly identified modifications are also involved in regulation of gene expression. By genome wide analysis (MeDIP-seq, 5hmC-IP-seq) we have identified genes/loci that are differentially methylated and hydroxymethylated in primary human HCCs. It is well established that epigenetic changes at certain loci are causally linked to tumorigenesis. Our goal is elucidate whether differential methylation/ hydroxymethylation at certain genomic regions alters their expression to promote HCC development.

My goal is to train graduate and undergraduate students and post-doctoral researchers in biomedical research, successfully get external funding to conduct continue research and publish high impact papers on liver cancer. I have mentored 5 graduate students, several post-doctoral fellows and undergraduate students and one high school student. Two graduate students I mentored received Pelotonia fellowship and 4 undergraduates received summer research fellowship. Currently, I am mentoring 4 graduate students and 2 post-doctoral fellows who are supported by NIH funding in HCC. I have received continuous funding from NIH in the last 15 years.