lncRNA-ENST00000564363抑制AMPK信号通路促非酒精性脂肪肝病脂质沉积的机制研究

Nonalcoholic fatty liver disease（NAFLD) is a kind of chronic liver disease， whose pathogenesis is unclear yet. lncRNA is the regulatory factor critical to lipid metabolism and is suggested to be a new therapeutic target. At the early stage, applicant filtered different IncRNA from humane NAFLD and normal liver via chip-gene and found out that IncRNA ENST00000564363 presented abnormal high expression in NAFLD.With further research, ENST00000564363 was proved to be able to degrade the expression of AMPK and facilitate deposit of intracellular lipid.Considering the significant regulatory effect of AMPK signal channel in lipid metabolism, research group put forward the scientific idea that ENST00000564363 takes part in the occurrence of NAFLD by controlling AMPK signal channel . The research object is mouse model raised by HFD (high fat diet) along with cell model induced by oleic acid. The research is based on molecular biology approach, such as qRT-PCR, Western blot, RNA interference to explore the influence and molecular mechanisms of ENST00000564363 over NAFLD Lipidoses in order to provide brand new idea for prevention and treatment.

非酒精性脂肪肝病（NAFLD）是常见的慢性肝病，其发病机制尚不明确。lncRNA是脂质代谢的重要调节因子，有望成为一种新的治疗靶点。申请人前期利用基因芯片对人NAFLD病理肝组织中差异表达的lncRNA进行筛选，发现lncRNA ENST00000564363在NAFLD组织中呈异常高表达。进一步研究发现ENST00000564363抑制AMPK的表达、促进细胞内脂质沉积。鉴于AMPK信号通路在脂质代谢中的重要调控作用，课题组提出以下科学设想：ENST00000564363通过AMPK信号通路促进脂质沉积，参与NAFLD的发生。本研究拟以油酸诱导的L02细胞模型及高脂饲料诱导的小鼠模型为研究对象，运用qRT-PCR、western blot和RNA干扰等实验方法探讨ENST00000564363对NAFLD脂质沉积的影响及分子机制，以期为NAFLD的防治提供新的思路。

非酒精性脂肪性肝病（Non-alcoholic fatty liver disease，NAFLD）是常见的慢性肝病，发病机制不明导致目前临床上缺乏针对该疾病的靶向治疗药物。申请人前期提出lncRNA-ENST00000564363可抑制AMPK信号通路促进肝细胞内脂质沉积，进而诱导NAFLD的发生。本项目分别通过油酸（OA）诱导人正常肝细胞构建的NAFLD细胞模型和高脂饮食构建的NAFLD大鼠模型，在体外及体内水平均证实了lncRNA ENST00000564363促进肝细胞脂质沉积的作用。进一步运用RNAi技术、拮抗剂干预、慢病毒感染等多个技术手段，证实ENST00000564363发挥作用的分子机制，即ENST0000056436可抑制AMPK表达，减少SREBP-1c磷酸化，上调下游脂质合成相关基因表达，促进脂质合成和沉积，参与NAFLD发生的调控。在完成项目预期研究目标的基础上，又通过高通量芯片对NAFLD患者体内lncRNA和mRNA表达谱进行分析，结果显示与正常肝组织相比，NAFLD肝组织中535个lncRNAs和760个mRNAs上调，1200个lncRNAs和725个mRNAs下调。这些异常表达的lncRNAs可能与NAFLD的发生发展有关。最后，对lncRNA CCAT1这一特定lncRNA在NAFLD中的分子生物学功能及机制进行查证后发现，lncRNA CCAT1通过与LXRα互为miR-613的ceRNA，促进NAFLD患者肝细胞脂质的合成。这些研究结果表明lncRNA在NAFLD发病中具有关键作用，相关数据也为今后系统分析lncRNAs在NAFLD中的生物学功能及机制奠定了理论基础，同时，为NAFLD的诊断和新型靶向药物的研制提供新的思路。