长链非编码RNA Gm26584 调控肝脏胆固醇代谢的作用及机制研究

As a precursor of steroids and bile acids, cholesterol plays an important role in vivo. It is of great significance to maintain cholesterol homeostasis in the prevention and treatment of atherosclerotic diseases caused by hypercholesterolemia. Recent studies indicate that the role of long chain non coding RNA (LncRNA) on regulating hepatic lipid metabolism has became more important. The scientific hypothesis of this project is: Accumulation of hepatic lipid altered the expression level of LncRNA, and interacts with their target proteins, regulates the expression level of genes of the lipid metabolism thereby regulating the hepatic lipid metabolism. We identified 16 LncRNAs which expression level significantly different in fatty liver samples of NAFLD mice compared with control mice by RNAseq. Pathway analysis shows that these LncRNAs strongly associated with lipid metabolism. Our preliminary results showed that the specific knockdown of Gm26584 in liver, one of the LncRNAs we identified from RNAseq, can significantly reduce the content of cholesterol in blood and liver, and significantly increase the mRNA level of LDLR and CYP7A1, indicating that Gm26584 is closely related to cholesterol metabolism. The proposed study will 1) determine the transcriptional regulation of Gm26584 2) explore the physiological role of Gm26584 in regulating hepatic cholesterol metabolism 3) find interacting protein of Gm26584, combine the in vivo and in vitro data elucidate the molecular mechanism of Gm26584 on regulating hepatic cholesterol metabolism. To provide new theoretical basis for prevention and treatment of atherosclerosis and cardiovascular diseases caused by hypercholesterolemia.

维持体内胆固醇代谢稳态对于预防和治疗高胆固醇血症导致的动脉粥样硬化性疾病具有重要意义。近年长链非编码RNA(LncRNA)在肝脏脂肪代谢方面的作用日益受到关注。本项目的科学假设为：肝脏脂肪积累，改变LncRNA的表达水平，并通过与靶蛋白相互作用，调节脂肪代谢通路基因的表达，从而调控肝脏的脂肪代谢。本课题组的预实验结果显示：通过RNAseq筛选出16个在脂肪肝中表达水平显著改变并可能和脂肪代谢相关的LncRNAs。将其中的Gm26584在肝脏中特异性敲降后可以显著降低血液和肝脏中胆固醇的含量，并显著提高肝脏胆固醇代谢通路中重要基因LDL受体和CYP7A1 mRNA的表达水平，预示其与胆固醇代谢密切相关。本项目拟在前期工作基础上，研究其转录表达调控、在肝脏中胆固醇代谢中的作用、寻找相互作用蛋白或靶蛋白、最终揭示其调节肝脏胆固醇代谢的分子机制。为预防和治疗动脉粥样硬化性疾病提供新的理论依据。

维持体内甘油三酯和胆固醇代谢稳态对于预防和治疗高脂血症导致的动脉粥样硬化性疾病具有重要意义。近年来长链非编码RNA(lncRNA )在肝脏脂肪代谢方面的作用日益受到关注。我们知道在肝脏脂代谢紊乱疾病情况下，肝脏脂肪积累，血脂升高，可以改变肝脏中lncRNAs的表达水平。这些lncRNAs再通过与其靶蛋白相互作用，调节脂肪代谢通路基因的表达，从而调控肝脏的脂肪代谢。因此本项目在高脂饮食诱导高脂血症的小鼠肝脏中寻找与肝脏脂质代谢相关的lncRNAs并探讨其分子机制，为预防和治疗高脂血症提供新的理论依据和药物靶点。我们首先通过高脂饮食诱导建立高脂血症的小鼠模型，然后，利用RNA-seq 分析比较了正常饮食和高脂饮食23 周后小鼠肝脏组织中的lncRNAs，筛选出表达水平较高，高脂血症中显著改变，可能和脂肪代谢相关的lncRNAs。我们从这些变化的lncRNAs中鉴定出lncRHL是一个受到高脂饮食负调控，主要分布于肝脏组织的功能未知的基因间lncRNA。我们发现在肝脏中特异性敲降lncRHL后可以显著升高血液中甘油三酯和胆固醇的含量，降低肝脏中甘油三酯和胆固醇的含量。进一步的研究发现在肝脏中特异性敲降lncRHL可以显著降低与VLDL分泌负相关的重要基因Bmal1，SHP的表达，促进MTTP的表达，从而促进肝脏VLDL的分泌。此外在肝脏中特异性敲降lncRHL后可以降低胆固醇转运通路中关键基因GATA4，ABCG5，ABCG8的表达水平。而在肝脏特异性过表达lncRHL后，可以显著升高Bmal1基因的表达水平，降低MTTP的表达，从而抑制肝脏VLDL分泌。这些结果提示lncRHL与肝脏的VLDL代谢密切相关。随后通过进一步的RNA pull down和RIP实验，我们发现lncRHL可以和hnRNPU直接结合并稳定hnRNPU的蛋白表达水平从而调控hnRNPU的下游靶基因Bmal1，SHP和MTTP的表达水平进而调节肝脏的VLDL分泌。结论：lncRHL 是一个新型的调控肝脏脂质代谢的lncRNA，lncRHL通过与hnRNPU直接结合稳定hnRNPU的蛋白水平，进而调节hnRNPU下游BMAL1-SHP-MTTP通路调控VLDL的分泌，同时通过Bmal1-GATA4-ABCG5/ABCG8通路调控胆固醇的代谢，为高脂血症及NAFLD的防治提供了新策略和理论依据。