microRNA-185介导的胆固醇代谢调控机制研究

Atherosclerosis, the main cause of cardiovascular disease, is associated closely with lipid disorders. Both LDL receptor (LDLR) pathway and reverse cholesterol transport (RCT) pathway mediated by HDL play an important role in lipid metabolism. So far, LDLR regulation remains the main target of anti-atherosclerosis drug discovery. MicroRNAs (miRNAs) are hairpin-derived RNAs of about 20-24 nucleotides long, which posttranscriptionally repress the expression of target genes usually by binding to the 3'UTR of their mRNAs. One miRNA may regulate several genes, while one single gene may be regulated by several miRNAs at the same time, which is known as "multi-targets, fine tuning, functional redundancy" properties of miRNA regulatory mechanism, implicating a complex and highly orchestrated mode of gene regulation. In our previous study, miR-185 was found to regulate the expression of HDL receptor (SR-BI) and SR-BI mediated HDL-C selective uptake in RCT pathway. The following research indicated that the plasma total and LDL cholesterol of ApoE-KO mice were both decreased by miR-185 inhibitor (anti-miR-185). Then we found that miR-185 regulated the expression of LDLR, as well as some transcriptional factors (TFs) and RNA binding proteins (RBPs) relative to LDLR regulation. Based on this, we are going to further investigate the regulatory mechanism of miR-185 on LDLR expression by directly binding to its 3'UTR, and explore miR-185 mediated-TFs or RBPs involved cholesterol regulatory network. The results obtained in this study may provide new insights into the regulatory mechanism of lipid metabolism, and furthermore, potential novel targets for lipid-regulating agent discovery.

动脉粥样硬化是心血管疾病的主要病理基础，与脂质代谢紊乱密切相关，LDL受体（LDLR）途径和HDL介导的胆固醇逆转运（RCT）途径在脂代谢过程中发挥着重要作用。由于"多靶、微调、功能冗余"等特性，miRNA在基因调控网络中发挥了复杂而精细的调节作用。本课题组前期工作中发现miR-185可调控HDL受体SR-BI表达及其在RCT中的功能，进而发现miR-185抑制剂可降低小鼠血浆总胆固醇及LDL胆固醇水平，并初步证实miR-185对LDLR以及其调控相关的转录因子（TFs）和RNA结合蛋白（RBPs）的调节作用。本项目拟在分子、细胞及动物水平，继续探究miR-185对LDLR表达的直接调节作用及机制，并通过研究miR-185与TFs或RBPs组成的调控单元，解析miR-185介导的脂代谢综合调控网络，研究结果将可能为新型调血脂药物的研发提供新的靶点和思路，具有重要的科学意义和应用价值。

动脉粥样硬化是心血管疾病的主要病理基础，与脂质代谢紊乱密切相关，低密度脂蛋白受体（LDLR）途径和高密度脂蛋白（HDL）介导的胆固醇逆转运（RCT）途径在脂代谢过程中发挥着重要作用。由于"多靶、微调、功能冗余"等特性，miRNA在基因调控网络中发挥了复杂而精细的调节作用。本课题组前期工作中发现miR-185可调控HDL受体SR-BI表达及其在RCT中的功能，本工作发现miR-185还可参与调控LDLR的表达及其功能。LDL是携带胆固醇最多的脂蛋白，主要功能是将血浆中的胆固醇运输到身体各组织细胞，参与细胞膜及各类激素的形成，多余的胆固醇则运输到肝脏合成胆汁酸。当LDL过量时会被氧化形成氧化型LDL（oxLDL），其携带的胆固醇在外周血管内壁沉积，导致动脉粥样硬化的发生。血浆中绝大多数LDL-C的清除依赖于LDLR途径，而肝脏是LDL-C代谢的主要器官，因此肝脏LDLR表达水平决定了血浆中的LDL-C水平。本项目工作发现miR-185靶向于LDLR 3’-UTR并抑制人肝癌细胞HepG2中LDLR的表达；但意外的是，miR-185抑制剂对LDLR表达同样有抑制作用。我们经过进一步研究发现，LDLR转录后调节元件KSRP也是miR-185的直接靶点，其表达受到miR-185的调控；KSRP基因沉默消除了miR-185抑制剂对LDLR表达的不正常抑制现象，并使其恢复对LDLR表达的上调。这表明，miR-185除了直接靶向LDLR，还通过KSRP间接调控LDLR表达。miR-185、KSRP和LDLR形成一种不一致前馈回路（feedforward loop，FFL），miR-185通过这一网络模体实现对LDLR表达的微调，并维持血浆胆固醇的动态平衡。此外，miR-185还可通过调节cyp7a1的表达，在胆汁酸代谢中也发挥了重要作用。动物水平结果显示，miR-185抑制剂可显著抑制血浆总胆固醇及LDL胆固醇的含量，并且可显著降低主动脉斑块的形成以及脂质在心脏流出道的堆积，展现出良好的抗动脉粥样活性。进一步我们利用纳米脂质体包载miR-185抑制剂以寻找更适合的给药方式。通过研究解析miR-185介导的脂代谢综合调控网络，研究结果将可能为新型调血脂药物的研发提供新的靶点和思路，具有重要的科学意义和应用价值，而miR-185抑制剂则有望发展为新型抗动脉粥样硬化核酸药物候选物。