长链非编码RNA APOA4-AS在肥胖小鼠肝脏脂质代谢中的作用机制研究

Dysregulated lipid homeostasis in the liver is closely associated with obesity. Whether lncRNA is invovled in obesity induced dysregulation of lipid homeostasis in the liver is largely unknown. Our hypothesis is that in obesity, abnormal lipid accumulation induces lncRNA expression, which increases the expression of lipid metabolism-related genes and finally induces fatty liver disease and hypertriglyceridemia by directly binding to some mRNA, genome DNA or proteins. In our preliminary studies, we identified 15 lncRNA which were abnormally expressed in the liver of db/db obesity mice model. Gene enrichment (GO) analysis shows that these lncRNAs neighboring genes are associated with lipid metabolism. One of these lncRNAs GM10680 was named as APOA4-AS because it is transcribed from the opposite strand of APOA4 gene locus. Both APOA4-AS and APOA4 expression are abnormally elevated in obesity. APOA4-AS specifically regulates APOA4 expression. APOA4-AS transcript directly binds to mRNA stabilizing protein HuR, and deletion of HuR reduces both APOA4-AS and APOA4 transcript levels. In this proposal, we will further elucidate the mechansims of APOA4-AS in the regulation of fatty liver disease and lipid metabolism in the obesity. This study will provide a new drug target for the treatment of type 2 diabetes.

肥胖导致肝脏脂质代谢异常。长链非编码RNA（LncRNA）是否参与了肝脏脂质代谢紊乱还不清楚。本项目的科学假设为：肥胖引起脂肪积累，诱导lncRNA的表达，lncRNA与mRNA、基因组DNA或某些蛋白结合，调节脂质代谢相关基因表达，进一步引起脂肪肝和高血脂。本课题组已筛选出15个lncRNA在肥胖db/db小鼠中异常表达，这些lncRNA附近的基因均与脂质代谢有关，其中一个lncRNA GM10680(现命名为 APOA4-AS)在肥胖小鼠肝脏中异常高表达，APOA4-AS正反馈调节了APOA4的表达，并且直接结合RNA稳定蛋白HuR。本项目拟在前期工作基础上，通过肝脏组织特异性APOA4-AS敲除或过表达、lncRNA-mRNA相互作用分析、lncRNA-蛋白相互作用分析、RNA稳定性分析等手段，研究APOA4-AS在肥胖诱导的脂肪肝和脂质代谢机制，为2型糖尿病的诊治提供理论依据。

肥胖状态下肝脏糖脂代谢稳态失衡，其分子机制并不清楚。在本项目的支持下，本课题组阐明了APOA4-AS、GM2788和Purβ在肥胖小鼠肝脏糖脂代谢稳态失衡中的作用机制。RNA-seq分析筛选出db/db肥胖小鼠肝脏中异常表达的长链非编码RNA，其中包括APOA4-AS和GM2788。我们确定了这2个lncRNA的序列特征：1）APOA4-AS转录本共有909nt，并且含有一段由内含子表达序列，大约207nt；2）GM2788-002转录本共有893nt，并且3’末端带有poly A的尾巴，证实RACE实验已经扩增到了末端,与Ensembl数据库提供的序列相比3’端多出96bp。我们深入研究了APOA4-AS和GM2788调节糖脂代谢的分子机制：1）APOA4-AS通过调节APOA4的表达进而影响了血液中的甘油三酯水平，RNA结合蛋白HuR参与这一过程；2）GM2788及其结合蛋白Purβ调节肝脏糖代谢：饥饿和肥胖状态下，Purβ表达增加，可以结合Adcy6启动子，促进Adcy6表达，进而促进Glucagon/cAMP/PKA/p-CREB信号通路，促进糖异生，与之相对应，肝脏特异敲低Purβ可以抑制Adcy6的表达，抑制Glucagon/cAMP/PKA/p-CREB信号通路，抑制糖异生，降低肥胖小鼠血糖。在本项目的支持下，我们还阐明了NIK在胰岛β/α细胞中的重要作用机制，阐明了METTL3在能量代谢和胰岛素分泌中的作用机制。这些重要发现揭示了肥胖状态下糖脂代谢稳态失衡的新机制，提供了新的药物靶点。