Circulating microRNAs Control Cholesterol Homeostasis through hepatic mechanisms

描述（由申请人提供）：肝脏通过调节脂蛋白摄取和分泌以及胆固醇生物合成，在胆固醇体内平衡中发挥核心作用。MicroRNA（miRNA）是转录后控制基因表达的小的非编码调节RNA，并且已被证明调节肝脏胆固醇代谢。miRNA在所有细胞类型中表达并且存在于血浆中。人和小鼠脂蛋白转运特异性miRNA并将其递送至受体细胞，包括肝细胞。脂蛋白递送miRNA导致特异性靶mRNA水平的降低。脂蛋白相关的miRNAs，miR-223和miR-24，发现于肝脏中，并通过计算预测靶向胆固醇生物合成酶以及各种脂蛋白受体。体外研究表明，高密度脂蛋白（HDL）-miR-223通过清道夫受体BI（SR-BI）转移至肝细胞。基于这些初步的发现，我假设特定的循环miRNA通过肝脏内的功能靶向来控制胆固醇代谢。该提案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转移到肝脏中的贡献。为了确定转移途径，LDL-miRNA递送将在转移效率和亚细胞定位方面与HDL-miRNA递送区分开。为了定量细胞外miRNA在细胞内mRNA靶向复合物上的肝掺入，将使用高通量小RNA测序进行光活化核糖核苷增强的交联免疫沉淀。肝脏miR-223和miR-24靶向胆固醇生物合成酶和脂蛋白受体将通过体外功能丧失和过表达研究进行验证。将使用miR-223-/-、SR-BI-/-和LDLR-/-敲除小鼠在体内评估经验证的靶标。该提案将系统地表征脂蛋白-miRNA递送，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.