lnc-HC调控PPARγ参与NAFLD肝脏脂肪酸代谢的分子机制研究

From the rats with high-fat high-cholesterol diet induced nonalcoholic fatty liver disease (HFHCD-NAFLD), we identified a novel long noncoding RNA (lncRNA) lnc-HC, and clarified its molecular mechanism in regulating hepatocytic cholesterol metabolism both in vitro and in vivo. Owing to the complexity of hepatic lipid metabolism and close correlation between cholesterol and fatty acid metabolism, we focus on pinpointing lnc-HC roles in regulating hepatic fatty acid metabolism in this study. Our preliminary data have shown that free fatty acids (FFAs) treatment of rat hepatocyte cell line CBRH-7919 resulted in the increment of lnc-HC expression. In addition, lnc-HC could negatively regulate PPARγ expression and lipid content in the CBRH-7919 cells. We thus speculate that lnc-HC could control hepatic fatty acid metabolism through regulating PPARγ expression, and the molecular mechanism requires fully understanding. In this study, we plan to observe the effects of lnc-HC on hepatocytic fatty acid metabolism, by detecting the concentrations of FFAs and triglyceride (TG) in lnc-HC over-expression/knockdown CBRH-7919 cells, which are treated with FFAs, and also in cell culture medium. It has been well known that lncRNAs regulate gene expression through three major different mechanisms, including: 1) lncRNA binds with co-regulator protein to form an lncRNA-protein complex, and regulates gene expression through the function of co-regulator; 2) lncRNA competes with target mRNA to bind regulatory miRNAs, like sponge, which is called as competing endogenous RNA (ceRNA); 3) lncRNA directly binds target mRNA to form an lncRNA-mRNA complex, and regulates gene expression through affecting mRNA stability. According to these three classical mechanisms, we verify the molecular mechanism of lnc-HC in regulating hepatocytic fatty acid metabolism through affecting PPARγ expression, by using the informatics analysis, RNA pull-down and RIP technologies. Finally, we detect the indexes related to fatty acid metabolism in livers of the high-fat diet induced NAFLD rats, which are intervened with lnc-HC shRNA adenovirus. The results will offer experimental evidence to clarify the precise molecular mechanism of lnc-HC in regulating hepatic fatty acid metabolism. The findings also provide novel insights into understanding pathogenesis of lipid metabolism disorders and NAFLD.

我们前期发现长链非编码RNA lnc-HC在大鼠高脂肝细胞模型中显著高表达，且负向调控PPARγ的表达及肝细胞脂滴形成，推测lnc-HC通过调控PPARγ通路抑制肝脏脂肪酸代谢。为阐明其分子机制，拟采用混合脂肪酸处理lnc-HC稳定高/低表达肝细胞，通过检测细胞及培养液中游离脂肪酸、甘油三酯含量，观察lnc-HC对肝细胞脂肪酸代谢表型的影响；在lnc-HC稳定高/低表达肝细胞中，检测PPARγ通路关键分子的表达，明晰lnc-HC参与的脂肪酸代谢通路；采用生物信息学、RNA pull-down、RIP等技术，诠释lnc-HC调控PPARγ基因表达、参与肝细胞脂肪酸代谢的分子机制；最后利用高脂饮食诱导大鼠NAFLD，腺病毒干扰lnc-HC表达，观察大鼠血清和肝脏代谢指标，验证lnc-HC对NAFLD肝脏脂肪酸代谢的作用。结果将丰富lncRNA对脂代谢作用的认识，为NAFLD的控制提供新思路。

众所周知，非酒精性脂肪肝是以肝细胞脂肪异常蓄积为临床表征的慢性疾病。代谢性核受体作为基因表达的上游调控者，在维持脂质代谢稳态中起到至关重要的作用。我们前期的研究中，一例全新的长链非编码RNA（大鼠lnc-HC）被成功鉴定，并发现该基因通过负性调节Cyp7a1和Abca1两个基因的表达参与了肝细胞胆固醇代谢调节过程。在该基金的资助下，我们进一步研究了lnc-HC对肝脏脂肪酸和甘油三酯代谢的作用和调节机制。首先，通过lnc-HC稳定干扰细胞系的鉴定，我们发现其能够负性调节代谢性核受体PPARγ的表达，而PPARγ是参与肝细胞脂肪酸代谢的重要转录因子之一。接着，细胞实验表明lnc-HC通过负性调节PPARγ的表达而抑制肝细胞脂滴形成。进一步，我们探索了其中的分子机制：miR-130b-3p能够靶向PPARγ 3'UTR从而负性调节其mRNA和蛋白质表达水平，而lnc-HC通过正向调节miR-130b-3p的转录及加工成熟过程从而间接抑制了PPARγ。值得注意的是，lnc-HC对miR-130b-3p的转录修饰调节与传统的内源性竞争性RNA机制不同，是一种新的调节模式。此外，在体动物模型结果显示，lnc-HC的敲低显著下调了miR-130b-3p并促进PPARγ的表达水平；动物表型表现为肝脏甘油三酯积聚及血脂异常。该结果帮助我们进一步了解了lnc-HC在肝脏脂质代谢中的调控作用，同时也为脂质代谢紊乱及非酒精性脂肪肝等疾病的检查和治疗提供了潜在的新靶点。