Alcohol abuse has been attributed to 3.2% of the world's disease burden. Chronic abuse manifests as reversible steatosis, steatohepatitis and/or irreversible cirrhosis. Regardless abstinence, 5-15% of patients with steatohepatitis still progress to cirrhosis and hepatocellular carcinoma (HCC). Studies have demonstrated the role of miR-122 as a mediator of hepatic metabolism, cellular differentiation and the development of HCC. Our laboratory has shown decreased miR-122 in a four-week chronic-alcohol mouse model. The role of miR-122 in ALD is unknown. Bioinformatic miRNA target prediction tools suggest Hypoxia-Inducible Factor 1-? (HIF1?) is a primary target of miR-122. HIF1? is critical to the progression of ALD.
Specific Aim 1 will study the role of miR-122 in the pathogenesis of chronic alcohol-induced hepatitis. C57Bl/6 mice will be transfected using a hepatocyte-tropic adeno-associated virus serotype 8 (rAAV) vector to either knockdown or overexpress miR-122 via tough decoy (TuD) or the miR-122 respectively. Mice will be maintained on Lieber-DeCarli diet for 28 days. On day 28 mice will be withdrawn from alcohol and given 150- mg/kg acetaminophen (APAP). Mice will be sacrificed on day 30 to assess regeneration. Livers sections will be analyzed histologically for morphological changes, lipid accumulation, and regeneration. In addition, we will determine the impact on miR-122 modulation on hepatic inflammation and differentiation through expression analysis of inflammatory cytokines and cell cycle regulators associated with ALD. MiR-122 has been correlated with a network of Liver Enriched Transcription Factors (LETFs). Collectively, these LETFs function as master regulators of hepatic function. HNF6?, specifically, has been shown to function in a positive feedback loop with miR-122.
Specific Aim 2 will examine the effect of miR-122, its overexpression and knockdown, on HNF6? in a chronic-alcohol model. We will also examine if miR-122 can reduce ALD through enhancement of HNF6? and the LETF network. The rAAV8 vector stated above will deliver anti- HNF6? shRNA or rHNF6 in vivo. The mice will be treated and assayed as described above. During the first year of this fellowship we aim to examine the effect of miR-122 on the severity of ALD development using gene therapy. The remaining two years of my Ph.D. (funding yrs 2 &3) will be spent studying the effect miR-122 and HNF6? regulation of the LETF network in the progression of ALD. During years 4 and 5 of funding I shall complete my M.D. training while finalizing any work required for publication. Collectively, we propose to examine two potential avenues by which miR-122 may serve as a potential therapeutic modality. First, is the development of steatosis though inhibition of HIF1? and secondly, is the regeneration after alcohol and APAP-induced liver injury.
This application aims to explore the biological mechanisms that contribute to alcoholic liver disease and the development of liver cirrhosis. The experiments will explore the use of gene therapy to restore hepatic function to prevent alcohol and toxin-induced liver damage. A greater understanding of these processes will allow for better treatment, prevention and prognosis of alcoholic liver disease.