Circulating microRNAs Control Cholesterol Homeostasis through hepatic mechanisms

描述(申请人提供)：肝脏通过调节脂蛋白的摄取和分泌，以及胆固醇的生物合成，在胆固醇稳态中发挥核心作用。MicroRNAs(MiRNA)是一种小的非编码调控RNA，它在转录后控制基因的表达，并已被证明调节肝脏的胆固醇代谢。MiRNAs在所有类型的细胞中都有表达，并存在于胞浆中。人和鼠脂蛋白运输特定的miRNAs，并将它们运送到包括肝细胞在内的受体细胞。脂蛋白递送miRNAs导致特异性靶基因水平的降低。脂蛋白相关的miRNAs，miR-223和miR-24，在肝脏中被发现，并被计算预测以胆固醇生物合成酶和各种脂蛋白受体为靶标。体外研究表明，高密度脂蛋白-miR-223通过清道夫受体BI(SR-BI)转移到肝细胞。基于这些初步发现，我假设特定的循环miRNAs通过肝脏内的功能靶向控制胆固醇代谢。本研究旨在确定脂蛋白受体SR-BI和低密度脂蛋白受体(LDLR)在体内将脂蛋白结合的miR-223转移到肝脏中的作用。为了确定转运途径，将在转运效率和亚细胞定位方面区分低密度脂蛋白-miRNA和高密度脂蛋白-miRNA。为了定量肝细胞外miRNAs对细胞内信使核糖核酸靶向复合体的掺入，将用高通量小RNA测序进行光敏化-核糖核苷增强的交联型免疫沉淀。肝脏miR-223和miR-24靶向胆固醇生物合成酶和脂蛋白受体的研究将通过体外功能丧失和过度表达研究来验证。验证的靶点将在体内用miR-223-/-、SR-BI-/-和LDLR-/-基因敲除小鼠进行评估。这项建议将系统地表征脂蛋白-miRNA的传递，并研究其在肝脏基因调控和胆固醇稳态中的作用。

DESCRIPTION (provided by applicant): The liver plays a central role in cholesterol homeostasis through modulation of lipoprotein uptake and secretion, and cholesterol biosynthesis. MicroRNAs (miRNA) are small non-coding regulatory RNAs that post- transcriptionally control gene expression and have been demonstrated to regulate hepatic cholesterol metabolism. miRNAs are expressed in all cell-types and are present in plasma. Human and mouse lipoproteins transport specific miRNAs and deliver them to recipient cells, including hepatocytes. Lipoprotein delivery of miRNAs resulted in the reduction of specific target mRNA levels. Lipoprotein-associated miRNAs, miR-223 and miR-24, are found in liver and are computationally predicted to target cholesterol biosynthetic enzymes, as well as various lipoprotein receptors. In vitro studies have revealed that high-density lipoprotein (HDL)-miR-223 is transferred to hepatocytes by the scavenger receptor BI (SR-BI). Based on these preliminary findings, I hypothesize that specific circulating miRNAs control cholesterol metabolism through functional targeting within the liver. This proposal aims to determine the contribution of lipoprotein receptors, SR-BI and low-density lipoprotein receptor (LDLR), in transferring lipoprotein-bound miR-223 to the liver in vivo. To determine the transfer pathway, LDL-miRNA delivery will be differentiated from HDL-miRNA delivery in transfer efficiency and sub-cellular localization. To quantify the hepatic incorporation of extracellular miRNAs onto intracellular mRNA targeting complexes, photoativatable-ribonucleoside-enhanced crosslinking immunopreciptation will be performed with high-throughput small RNA sequencing. Hepatic miR-223 and miR-24 targeting of cholesterol biosynthetic enzymes and lipoprotein receptors will be validated by loss-of-function and overexpression studies in vitro. Validated targets will be assessed in vivo with miR-223-/-, SR-BI-/-, and LDLR-/- knockout mice. This proposal will systematically characterize lipoprotein-miRNA delivery and examine its role in hepatic gene regulation and cholesterol homeostasis.