Control of Sterol and Lipoprotein Homeostasis by miRNA

描述（由申请人提供）：动脉粥样硬化是心脏病发作、中风和外周动脉疾病的主要原因，这三种疾病加起来占美国所有死亡人数的30%。多项研究证实胆固醇稳态异常是动脉粥样硬化发生的危险因素。我们和其他人最近报道了miR-33，这是一种在SREBP-2的内含子16中编码的保守microRNA。在接受他汀类药物治疗的高胆固醇血症患者中，SREBP-2的临床重要性被揭示，SREBP-2通过增加肝脏中SREBP-2及其靶ldl -受体的表达来降低ldl -胆固醇水平。我们发表的和初步的研究表明，miR-33的生理靶点包括ABCA1和ATP8B1。ABCA1是HDL脂化和逆向胆固醇转运的关键转运蛋白，ATP8B1是与肝内胆汁淤积相关的磷脂翻转酶。该提案的总体目标是确定miR-33在胆固醇、胆汁和脂蛋白稳态中的作用。我们假设miR-33控制着固醇动员、胆汁排泄和HDL代谢的关键方面。重要的是，由于他汀类药物通常用于高胆固醇血症患者，他汀类药物诱导miR-33的表达，我们推测他汀类药物治疗的患者miR-33水平升高，miR-33持续下调ABCA1和ATP8B1。为了解决这些问题，我们提出以下3个具体目标：1)通过测量miR-33过表达或沉默对脂蛋白分泌和胆汁排泄的影响，确定miR-33在原代肝细胞和小鼠肝固醇稳态中的作用；2)通过体内实验，验证巨噬细胞miR-33表达改变导致胆固醇逆向转运和动脉粥样硬化病变大小改变的假设，确定巨噬细胞miR-33表达在Ldlr小鼠动脉粥样硬化病变胆固醇-/-动员和进展中的作用；3)通过表征miR-33系统沉默后产生的HDL的组成和生物学特性，以及评估抗miR-33寡核苷酸是否与他汀类药物在ApoE*3Leiden W hCETP小鼠中的动脉粥样硬化保护作用协同作用，验证体内系统沉默miR-33具有动脉粥样硬化保护作用的假设。胆固醇稳态、胆固醇稳态和动脉粥样硬化的多个方面仍不清楚。我们的数据表明，胆固醇- mir -33轴调节肝细胞和巨噬细胞生物学的关键方面。我们预计我们的研究将为控制细胞内胆固醇水平、胆汁排泄和循环脂蛋白的复杂调节网络提供新的线索。如果我们的假设是正确的，miR-33可能是治疗血脂异常和/或胆汁淤积的新疗法的靶点。！

DESCRIPTION (provided by applicant): Atherosclerosis is the primary cause of heart attack, stroke, and peripheral artery disease, which collectively account for >30% of all deaths in the US. Multiple studies recognized abnormal cholesterol homeostasis as a risk factor for the development of atherosclerosis. We and others have recently reported on miR-33, a conserved microRNA that is encoded within intron 16 of SREBP-2. The clinical importance of SREBP-2 is revealed in hypercholesterolemic patients treated with statins, which reduce LDL-cholesterol levels by increasing hepatic expression of SREBP-2 and its target the LDL-receptor. Our published and preliminary studies show that physiological targets of miR-33 include ABCA1, a transporter critical for HDL lipidation and reverse cholesterol transport, and ATP8B1, a phospholipid flippase linked to intrahepatic cholestasis. The broad goal of this proposal is to determine the role of miR-33 on cholesterol, bile and lipoprotein homeostasis. We hypothesize that miR-33 controls key aspects of sterol mobilization, bile excretion and HDL metabolism. Importantly, since statins are usually prescribed to hypercholesterolemic patients, and statins induce the expression of miR-33, we speculate that statin-treated patients will have elevated levels of miR-33 and persistent down regulation of ABCA1 and ATP8B1 by miR-33. To address these questions, we propose the following 3 specific aims: 1) Determine the role of miR-33 in hepatic sterol homeostasis in primary hepatocytes and in mice, by measuring the effect of over expression or silencing of miR-33 on lipoprotein secretion and bile excretion; 2) Test the hypothesis that altered macrophage miR-33 expression results in changes in reverse cholesterol transport and atherosclerotic lesion size, by performing experiments in vivo to determine the role of macrophage miR-33 expression on cholesterol -/- mobilization and progression of atherosclerotic lesions in Ldlr mice ; and 3) Test the hypothesis that systemic silencing of miR-33 is atheroprotective in vivo, by characterizing the composition and biological properties of HDL generated following systemic silencing of miR-33, and by evaluating whether anti-miR-33 oligonucleotides synergize with the atheroprotective effects of statins in ApoE*3Leiden W hCETP mice. There are still multiple aspects of cholesterol homeostasis, cholestasis and atherogenesis that remain obscure. Our data suggest that the cholesterol-miR-33 axis modulates key aspects of hepatocyte and macrophage biology. We anticipate that our studies will provide new clues into the complex regulatory networks that control intracellular cholesterol levels, bile excretion and circulating lipoproteins. If our hypothesis is true, miR-33 might be a target for novel therapies to manage dyslipidemias and/or cholestasis. ! PUBLIC HEALTH RELEVANCE: We propose to characterize miR-33, which defines a novel pathway that controls cholesterol, bile and lipoprotein homeostasis. We hypothesize that miR-33 modulates the expression of key genes involved in different aspects of sterol metabolism. The results of these studies might lead to novel, improved ways to manage patients with hypercholesterolemia and/or patients with cholestasis. !