The Role of miR-122 in Alcoholic Liver Disease

描述(申请人提供)：酒精滥用已被归因于3.2%的世界疾病负担。慢性滥用表现为可逆性脂肪变性、脂肪性肝炎和/或不可逆转的肝硬变。无论禁食与否，5%-15%的脂肪性肝炎患者仍会进展为肝硬变和肝细胞癌。研究表明miR-122在肝脏代谢、细胞分化和肝细胞癌的发生发展过程中起着重要的调节作用。我们的实验室在四周的慢性酒精中毒小鼠模型中显示miR-122减少。MiR-122在ALD中的作用尚不清楚。生物信息学miRNA靶标预测工具表明，缺氧诱导因子1-�(HIF1�)是miR-122的主要靶标。HIF1�在肌萎缩侧索硬化症的发生发展中起关键作用。目的1研究miR-122在慢性酒精性肝炎发病机制中的作用。C57BL/6小鼠将用嗜肝细胞腺相关病毒血清8型(RAAV)载体分别通过TUD或miR-122敲除或过表达miR-122。小鼠将保持28天的Lieber-DeCarli饮食。第28天，小鼠戒断酒精，给予150 mg/kg的对乙酰氨基酚(APAP)。小鼠将在第30天被处死以评估再生情况。将对肝脏切片进行组织学分析，以了解形态变化、脂肪堆积和再生。此外，我们还将通过与ALD相关的炎性细胞因子和细胞周期调节因子的表达分析，确定miR-122对肝脏炎症和分化的调控作用。MIR-122与肝脏富集型转录因子(LETF)网络相关。总而言之，这些LETF是肝功能的主要调节因子。具体地说，HNF6�已被证明与miR-122一起在正反馈循环中发挥作用。《特定目标2》将在慢性酒精模型中检测miR-122的过度表达和基因敲除对HNF6�的影响。我们还将研究miR-122是否可以通过增强HNF6�和LETF网络来减少ALD。上述rAAV8载体将在体内携带抗HNF6shRNA或rHNF6�。这些小鼠将接受如上所述的治疗和检测。在这项研究的第一年，我们的目标是通过基因治疗来检验miR-122对ALD发展的严重程度的影响。我博士剩下的两年(资助2年和3年)将用于研究miR-122和HNF6�调节LETF网络在ALD进展中的作用。在第4年和第5年的资助期间，我将完成医学博士培训，同时完成出版所需的任何工作。总而言之，我们建议研究miR-122可能作为一种潜在治疗方式的两种潜在途径。首先是抑制HIF1�导致的脂肪变性，其次是酒精和APAP诱导的肝损伤后的再生。

DESCRIPTION (provided by applicant): 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 On day 28 mice will be withdrawn from alcohol and given 150- mg/kg acetaminophen (APAP).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.