Circulating microRNAs Control Cholesterol Homeostasis through hepatic mechanisms

循环 microRNA 通过肝脏机制控制胆固醇稳态

• 批准号: 8729680
• 负责人: Kasey C Vickers
• 金额: $ 24.86万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-13 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8729680
• 关键词: Atherosclerosis; Bioinformatics; Blood Circulation; Cardiovascular Diseases; Cause of Death; Cell Communication; Cells; Cholesterol; Cholesterol Homeostasis; Complex; Country; Cytoplasm; Data; Disease; Drug Targeting; Effectiveness; Enzymes; Foundations; Functional RNA; Future; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Heart Diseases; Hepatic; Hepatocyte; High Density Lipoproteins; Human; Immunoprecipitation; In Vitro; Knockout Mice; LDL Cholesterol Lipoproteins; Lipoprotein Binding; Lipoprotein Receptor; Lipoproteins; Liver; Low Density Lipoprotein Receptor; Low-Density Lipoproteins; Luciferases; Mediating; Messenger RNA; MicroRNAs; Mus; Pathway interactions; Plasma; Play; Prevention; RNA Sequences; Regulation; Regulator Genes; Regulatory Pathway; Reporter Genes; Research Personnel; Rest; Ribonucleosides; Role; SR-BI receptor; Small RNA; Testing; Therapeutic; Training; United States; base; cell type; cholesterol biosynthesis; crosslink; extracellular; high density lipoprotein receptor; hypercholesterolemia; in vivo; intercellular communication; loss of function; novel; overexpression; prevent; public health relevance; receptor mediated endocytosis; uptake

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.

描述（由申请人提供）：肝脏通过调节脂蛋白的摄取和分泌以及胆固醇生物合成在胆固醇稳态中发挥核心作用。 MicroRNA (miRNA) 是小型非编码调节 RNA，可在转录后控制基因表达，并已被证明可调节肝脏胆固醇代谢。 miRNA 在所有细胞类型中表达并存在于血浆中。人类和小鼠脂蛋白转运特定的 miRNA 并将其递送至受体细胞，包括肝细胞。 miRNA 的脂蛋白传递导致特定靶标 mRNA 水平的降低。脂蛋白相关 miRNA miR-223 和 miR-24 存在于肝脏中，经计算预测它们会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 递送区分开来。为了量化细胞外 miRNA 在肝脏中掺入细胞内 mRNA 靶向复合物的情况，将通过高通量小 RNA 测序进行光活化核糖核苷增强交联免疫沉淀。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度表达研究进行验证。将使用 miR-223-/-、SR-BI-/- 和 LDLR-/- 敲除小鼠体内评估已验证的靶标。该提案将系统地描述脂蛋白-miRNA 传递的特征，并检查其在肝脏基因调控和胆固醇稳态中的作用。