miR-378/378* promotes hepatic lipid accumulation and progression of NAFLD to NASH

描述（由申请人提供）：非酒精性脂肪性肝病（NAFLD）是最常见的肝脏疾病，估计在工业化国家的一般人群中患病率为20-30%。它与许多风险因素相关，包括II型糖尿病、肝细胞癌（HCC）、心血管疾病和高脂血症。microRNA（miRNAs）是一类新的天然存在的小的非编码RNA，已知其在许多代谢紊乱中起关键作用，部分地通过抑制靶基因的表达。现在已经确定，将特异性miRNA或antimiR引入患病细胞和组织中可以诱导有利的治疗反应。事实上，miR-122抑制剂已作为HCV感染的治疗剂进入III期临床试验。肝细胞是控制脂质代谢的主要细胞类型，也是NAFLD中脂质蓄积的主要部位。为了解决它们在NAFLD发展中的潜在作用，我们最初鉴定了在肝细胞中高度特异性表达的miRNA，并发现其中一种miR-378/378* 在维持高脂饮食（HFD）的小鼠肝脏中显著诱导。此外，通过抑制miR-378/378*，我们能够显著预防HFD处理的小鼠中的肝脏脂质积聚和高脂血症。生物信息学和验证研究显示，miR-378/378* 直接抑制NASH和血脂异常的两个看门人NRF 1（核呼吸因子1）和SORT 1（分拣蛋白1），并诱导NASH启动子TNFα的表达。这些发现使我们假设miR-378/378* 通过同时调节NRF 1、SORT 1和TNFα的表达促进肝脏脂质蓄积和NAFLD向NASH的进展。本项目的目的是确定miR-378/378* 与NRF、SORT 1和TNFα的串扰在调节肝脏脂质蓄积和NAFLD向NASH进展中的作用。本研究的长期目标是阐明NAFLD及其进展为NASH的潜在机制，并开发miRNA抑制剂作为这两种疾病的治疗剂。三个具体目标旨在验证我们的假设。具体而言，我们将（1）建立miR-378/378* 影响HFD处理小鼠肝脏脂质的功能获得性研究，并确定其抑制是否通过调节NRF 1和SORT 1阻止肝脏脂质蓄积;（2）确定miR-378/378* 在促进NAFLD向NASH进展中的作用，并阐明该过程的潜在机制;以及（3）评估来自具有NAFLD和NASH谱的患者的一组肝脏样品中miR-378/378*、SORT 1、NRF 1和TNFα的水平。我们的研究旨在建立miR-378/378* 作为调节肝脏脂质代谢和NAFLD向NASH进展的新途径。强有力的初步数据和逻辑合理的实验设计相结合，使该项目具有高度的可行性。这些结果将为miRNAs的生理作用和机制提供新的见解，以及它们对这两种肝脏疾病的潜在治疗应用。

DESCRIPTION (provided by applicant): Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder with a prevalence estimated to be in 20-30% of the general population within the industrialized world. It is associated with a number of risk factors, including, type II diabetes, hepatocellular carcinoma (HCC), cardiovascular disease, and hyperlipidemia. MicroRNAs (miRNAs) are a new class of naturally occurring small non-coding RNAs that are known to play critical roles in a number of metabolic disorders, in part, by inhibiting expression of target genes. It is now well established that the introduction of specific miRNAs, or antimiRs into diseased cells and tissues can induce favorable therapeutic responses. Indeed, miR-122 inhibitor has entered phase III clinical trials as a therapeutic agent for HCV infection. Hepatocytes are the major cell type that controls lipid metabolism and the primary site of lipid accumulation in NAFLD. To address their potential role in the development of NAFLD, we initially identified miRNAs that are highly and specifically expressed in hepatocytes and found that one of them, miR-378/378*, is significantly induced in the livers of mice maintained on a high fat diet (HFD). Further, by inhibiting miR-378/378* we were able to significantly prevent hepatic lipid accumulation and hyperlipidemia in HFD-treated mice. Bioinformatic and validation studies revealed that miR-378/378* directly inhibited NRF1 (nuclear respiratory factor 1) and SORT1 (Sortilin 1), two gatekeepers of NASH and dyslipidemia, and induced expression of TNFα, a promoter of NASH. These findings led us to hypothesize that miR-378/378* promotes hepatic lipid accumulation and the progression of NAFLD to NASH by simultaneously modulating expression of NRF1, SORT1 and TNFα. The objective of this project is to determine the roles of the crosstalk of miR-378/378* with NRF, SORT1 and TNFα in regulating hepatic lipid accumulation and the progression of NAFLD to NASH. The long-term goal of this study is to elucidate the underlying mechanism(s) of NAFLD and its progression to NASH, and develop miRNA inhibitors as therapeutic agents for both disorders. Three Specific Aims are designed to test our hypothesis. Specifically, we will (1) establish gain-of-function studies for miR-378/378* in affecting hepatic lipids in HFD-treated mice, and determine whether their inhibition prevents hepatic lipid accumulation via modulation of NRF1 and SORT1; (2) determine the role of miR-378/378* in promoting progression of NAFLD to NASH, and elucidate the underlying mechanisms of this process; and (3) evaluate the levels of miR-378/378*, SORT1, NRF1 and TNFα in a set of liver samples from patients with a spectrum of NAFLD and NASH. Our studies are designed to establish miR-378/378* as a new pathway for the regulation of hepatic lipid metabolism and the progression of NAFLD to NASH. The combination of strong preliminary data and a logical and rationally based experimental design make this project highly feasible. The results will provide novel insights into the physiological roles and mechanisms of miRNAs, in addition to their potential therapeutic application for both of these hepatic disorders.