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如肥胖、动脉粥样硬化和代谢综合征的危险因素。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。我们观察到miR-30 c诱导肝癌细胞中MTP mRNA降解并减少apoB分泌。 此外，它还能降低小鼠的血浆脂质和肝脂蛋白的产生。因此，我们假设miR-30 c靶向MTP，减少apoB分泌，降低血脂，并预防动脉粥样硬化。 目标1：我们将通过使用过表达和化学拮抗方法操纵其在肝脏中的水平来评估miR-30 c在降低高脂血症和动脉粥样硬化中的功效。首先，将在喂食西方饮食的雄性C57/B16小鼠中评估miR-30 c、抗miR-30 c和乱序miR（Scr）对高脂血症的影响。接下来，我们将评估miR-30 c在降低WHHL兔血浆脂质中的功效。 第二，为了检验miR-30 c减少动脉粥样硬化的假设，我们将在西方饮食喂养的Apoe-/-和LDLr-/-小鼠的肝脏中表达miR-30 c、抗miR-30 c和Scr。将进行机制实验以确定miR-30 c降低肝脂蛋白产生。 目的2：我们将使用原代肝细胞和小鼠评估miR-30 c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。我们推测，血浆脂质的降低是由于辅助共翻译降解增加导致apoB合成减少所致。其次，将计划实验以记录为什么尽管MTP表达较低，但miR-30 c表达与脂肪变性无关。我们假设miR-30 c减少肝脏脂质生物合成并避免脂肪变性。第三，我们将确定miR-30 c和MTP mRNA之间相互作用的机制。我们的假设是，在补充网站的相互作用是至关重要的下调MTP的miR-30 c。我们将（a）评估“补充序列”在MTP/miR-30 c相互作用中的重要性，（B）确定miR-30 c的补充序列中降低MTP水平所需的最佳碱基对，以及（c）评估在诱导MTP mRNA降解中种子和补充位点之间形成不对称环的需要。 最后，我们将对miR-30 c的作用方式以及参与降低高脂血症和动脉粥样硬化的机制有新的认识。这些研究可能表明，miR-30 c通过诱导MTP mRNA的转录后降解和新生apoB的共翻译降解来调节血浆含apoB的脂蛋白。有关miR-30 c和MTP之间导致MTP mRNA破坏的分子相互作用的新信息将被收集。如果得到证实，miR-30 c可能作为一种潜在的新的治疗剂来治疗高脂血症、动脉粥样硬化和其他相关的代谢紊乱。

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.