新的人lncRNA:LNC-HC调控胆固醇代谢致NASH肝细胞损伤的分子机制研究

Nonalcoholic steatohepatitis (NASH) is defined as the presence of hepatic steatosis and inflammation with hepatocyte injury (ballooning) with or without fibrosis. It is part of clinical metabolic disorder syndrome called nonalcoholic fatty liver disease and is the inevitable stage from simple fatty liver progressing to manifest cirrhosis or even to hepatocellular carcinoma. But, the detailed pathogenesis of NASH is still unknown. Recent research indicated that long non-coding RNA (lncRNA) and free cholesterol (FC) were closely associated with lipid metabolism and many related diseases. It has been explored that lncRNAs were involved in the biosynthesis and excretion of cholesterol as well as the conversion from cholesterol to bile acid. FC accumulation results in hepatocyte injury and triggers NASH mediated by membrane fluidity variation, function deviation of membrane protein, endoplasmic reticulum stress response or the toxicity of oxidized cholesterol. .In our previous work, a novel lncRNA termed as lnc-HC has been identified from the rat and proved that lnc-HC was strongly related with NASH. lnc-HC could bind with the mRNA of Cyp7a1 and Abca1, the key molecules for cholesterol metabolism, mediated by co-regulation protein hnRNPA2B1 to form lncRNA-protein-mRNA complex which negatively control the cholesterol catabolism. Further investigation showed that high concentration of cholesterol up-regulated the expression of lnc-HC and in vivo interference of lnc-HC could obviously alleviate dyslipidemia resulted from high cholesterol diet. The human homologous molecule of lnc-HC, LNC-HC, has been preliminarily identified in human. However, the function of LNC-HC and the molecular mechanism of LNC-HC involved in cholesterol metabolism and NASH still maintain obscure..In this project, we firstly intends to use Northern blotting and fusion protein technique to identify LNC-HC and analyze the relationship of the hepatic LNC-HC expression level and cholesterol metabolism and clinical manifestation of NASH by using clinical samples. And then, ITRAQ, GO & pathway and RNA pull-down techniques will be employed to screen and determine the target genes and metabolic pathway of LNC-HC involved in cholesterol metabolism and the molecular mechanism regulated by LNC-HC. Furthermore, we will clarify the correlation between the FC accumulation and the hepatocyte injury resulted from LNC-HC dysregulation using the hepatocyte lines overexpressing and deleting LNC-HC and FC-hepatocyte damage model. All the results obtained from this project will be verified in clinical samples from NASH patients. .The findings will provide new insights into the molecular mechanism of LNC-HC involving in cholesterol metabolism and the onset of NASH.

我们从大鼠NASH模型中发现了一个全新的lncRNA:lnc-HC，证实其参与胆固醇代谢调控，与实验性NASH发生密切相关，并发现人中存在lnc-HC的同源序列LNC-HC，但新的人LNC-HC参与胆固醇代谢调控及NASH发生的功能未知、机制不明。本项目拟先用生物信息学方法、融合蛋白编码实验及Northern blotting鉴定LNC-HC；分析临床肝组织样本中LNC-HC表达水平与肝游离胆固醇（FC）、NASH表型的相关性；经iTRAQ、GO和pathway分析, RNA pull-down, RIP等技术筛选、验证并确定LNC-HC调控胆固醇代谢的靶基因、通路及分子机制；用LNC-HC高表达/敲除细胞株及FC损伤模型明确LNC-HC致肝细胞损伤的机制；在临床样本中验证实验结果。最终阐明LNC-HC调控胆固醇代谢致NASH肝细胞损伤的作用及机制，为NASH防治提供坚实理论和实验基础。

项目背景：我们前期研究工作发现并鉴定了一个新大鼠lncRNA：lnc-HC，高浓度胆固醇可通过激活 LXRs 表达活化 C/EBPβ进而上调 lnc-HC， lnc-HC可通过形成“lnc-HC- hnRNPA2B1- Cyp7a1/Abca1 mRNA”复合物，负向调控胆固醇分解代谢关键分子 Cyp7a1 和 Abca1 表达；在体干扰lnc-HC能改善高胆固醇饮食造成的大鼠脂代谢紊乱；但其在体发挥功能的分子机制不清。另外，发现人中存在lnc-HC的同源序列LNC-HC，但其功能未知、在NASH发生发展中的作用及机制不明。本项目旨在阐明人和大鼠的lnc-HC在NASH发生发展中的作用及机制。.主要研究内容及重要结果：（1）本课题组利用CRISPR-Cas9技术构建并繁育获得lnc-HC敲除SD纯合子大鼠。利用此lnc-HC敲除大鼠，明确了lnc-HC在高脂/高脂高胆固醇饮食诱导的非酒精性脂肪性肝炎（HFD-NASH、HFHC-NASH）发生中发挥着非常关键的作用；另外，对模型鼠的肝脏组织进行转录组测序（RNA-seq），发现脂肪酸/甘油三酯代谢变化最为显著。（2）阐明lnc-HC通过一种新的lncRNA调节机制发挥其在肝脂肪酸代谢中的作用。 lnc-HC表达降低显著抑制miR-130b-3p表达，进而诱导PPARγ表达，促进肝细胞脂滴的形成，并增加大鼠肝脏中的甘油三酯的浓度。（3）明确lnc-HC在肝甘油三酯代谢通路中的分子机制。体外和体内实验表明，lnc-HC抑制/敲除可降低RNA结合蛋白YBX1的表达，导致pnpla3表达下降，机体分解甘油三酯能力减弱，引起脂质积累及NASH的发生。（4）初步明确lnc-HC敲除引起脂肪组织积累的分子机制。Lnc-HC敲除可促进前脂肪细胞分化相关基因vkorc1l1表达升高，引起脂肪组织积累，加重肥胖的发生，进而引起NASH。.上述结果表明，lnc-HC在NASH的发生中起着非常关键的作用，是防治NASH 的潜在靶点，最终阐明lnc-HC调控脂肪酸/甘油三酯代谢致NASH的作用及机制，为NASH防治提供坚实理论和实验基础。