Regulation of plasma lipids and atherosclerosis by miR-30c

描述(由申请人提供)： 高血脂是各种心血管和代谢紊乱的危险因素，如肥胖、动脉粥样硬化和代谢综合征。我们的长期目标是找到通过靶向脂蛋白组装来降低血脂的方法。微粒体甘油三酯转移蛋白(MTP)是脂蛋白组装的关键蛋白。我们观察到miR-30c诱导肝癌细胞MTP mRNA降解和apoB分泌减少。此外，它还能降低小鼠的血脂和肝脂蛋白的产生。因此，我们假设miR-30c靶向MTP，减少载脂蛋白B的分泌，降低血脂，预防动脉粥样硬化。目的：通过调节miR-30c在肝脏中的表达水平和化学拮抗作用，评价miR-30c在降低高脂血症和动脉粥样硬化中的作用。首先，将评估miR-30c、抗miR-30c和scrble miR(Scr)对喂饲西方饮食的雄性C57/BL6小鼠高脂血症的影响。接下来，我们将评估miR-30c对WHHL兔的降血脂作用。其次，为了验证miR-30c减少动脉粥样硬化的假设，我们将在喂养APOE-/-和LDLR-/-的西方饮食小鼠的肝脏中表达miR-30c、抗miR-30c和Scr。将进行机械性实验，以确定miR-30c降低肝脂蛋白产生。目的2：利用原代肝细胞和小鼠评价miR-30c如何降低MTP表达、apoB分泌和高脂血症。首先，我们将揭示导致低血脂的机制。我们假设，血脂的降低是由于共翻译降解增加导致载脂蛋白B合成减少所致。其次，将计划进行实验，以证明为什么尽管MTP表达较低，miR-30c的表达与脂肪变性无关。我们推测miR-30c可以减少肝脏脂肪的生物合成，避免脂肪变性。第三，我们将确定miR-30c与MTP mRNA相互作用的机制。我们的假设是，补充位点的相互作用在miR-30c下调MTP的过程中起关键作用。我们将(A)评估“补充序列”在MTP/miR-30c相互作用中的重要性，(B)确定miR-30c补充序列中降低MTP水平所需的最佳碱基对，以及(C)评估种子和补充位点之间是否需要在诱导MTP mRNA降解方面形成不对称环。最后，我们将对miR-30c的作用机制以及参与降高脂血症和动脉粥样硬化的机制有一个新的理解。这些研究可能表明miR-30c通过诱导MTP mRNA的转录后降解和新生的apoB的共翻译降解来调节含apoB的脂蛋白。有关miR-30c和MTP之间导致MTP mRNA破坏的分子相互作用的新信息将被获得。如果得到证实，miR-30c可能成为治疗高脂血症、动脉粥样硬化和其他相关代谢疾病的潜在新治疗剂。

DESCRIPTION (provided by applicant): High plasma lipids are risk factors for various cardiovascular and metabolic disorders such as obesity, atherosclerosis and metabolic syndrome. Our long term goal is to find ways to lower plasma lipids by targeting lipoprotein assembly. Microsomal triglyceride transfer protein (MTP) is critical for lipoprotein assembly. We observed that miR-30c induces MTP mRNA degradation and reduces apoB secretion in hepatoma cells. Further, it lowers plasma lipids and hepatic lipoprotein production in mice. Hence, we hypothesize that miR- 30c targets MTP, reduces apoB secretion, lowers plasma lipids, and prevents atherosclerosis. Aim 1: We will evaluate the efficacy of miR-30c in reducing hyperlipidemia and atherosclerosis by manipulating its levels in the livers using overexpression and chemical antagonism approaches. First, effect of miR-30c, anti-miR-30c, and scramble miR (Scr) on hyperlipidemia will be assessed in male C57/Bl6 mice fed Western diet. Next, we will evaluate the efficacy of miR-30c in reducing plasma lipids in WHHL rabbits. Second, to test the hypothesis that miR-30c reduces atherosclerosis we will express miR-30c, anti-miR-30c and Scr in the livers of Western diet fed Apoe-/- and LDLr-/- mice. Mechanistic experiments will be performed to ascertain that miR-30c lowers hepatic lipoprotein production. Aim 2: We will evaluate how miR-30c reduces MTP expression, apoB secretion and hyperlipidemia using primary hepatocytes and mice. First, we will uncover mechanisms that lead to low plasma lipids. We hypothesize that decreases in plasma lipids occur due to reductions in apoB synthesis secondary to increased co-translational degradation. Second, experiments will be planned to document why miR-30c expression is not associated with steatosis despite lower MTP expression. We hypothesize that miR-30c reduces hepatic lipid biosynthesis and avoids steatosis. Third, we will identify mechanisms involved in the interactions between miR-30c and MTP mRNA. Our hypothesis is that interactions at the supplementary site are critical in the down regulation of MTP by miR-30c. We will (a) evaluate the importance of "supplementary sequences" in MTP/miR-30c interactions, (b) determine optimum base pairs required in the supplementary sequence of miR-30c to reduce MTP levels, and (c) assess the need of an asymmetric loop formation between seed and supplementary sites in inducing MTP mRNA degradation. At the end, we will have a novel understanding about the mode of action of miR-30c, and mechanisms involved in the lowering of hyperlipidemia and atherosclerosis. These studies might show that miR-30c regulates plasma apoB-containing lipoproteins by inducing posttranscriptional degradation of MTP mRNA and co-translational degradation of nascent apoB. Novel information about the molecular interactions between miR- 30c and MTP that lead to MTP mRNA destruction will be garnered. If proven, miR-30c might serve as a potentially new therapeutic agent to treat hyperlipidemia, atherosclerosis and other related metabolic disorders.