养殖鱼类普遍存在由于脂肪过度蓄积而导致的肝脂质代谢紊乱问题。研究发现microRNAs参与了陆上动物脂代谢过程，但其在鱼类中的作用并不清楚。我们在前期研究中发现，草鱼miR-122/33与肝脂代谢关系密切，且在SREBP-1c 3’UTR区有潜在结合位点。在此基础上，本项目拟以成功建立的草鱼脂变肝细胞及活体模型为材料，通过构建miR-122/33超表达和干扰载体，采用Western blot、qRT-PCR及酶活性测定等技术手段，检测miR-122/33对脂代谢生化指标、关键基因表达及酶活性等的影响，研究miR-122/33在肝脂代谢中的功能；采用荧光素酶报告基因系统检测miR-122/33和SREBP-1c 3’UTR的靶标关系，以期阐释miR-122/33调控SREBP-1c介导的草鱼脂代谢的分子机制。研究结果可为提高草鱼脂肪利用能力和预防脂肪肝等代谢综合症的发生提供新的科学依据。

Lipid metabolism disorders are frequently found often occurred in the fishes feeding with nutritionally -imbalanced diets. Many studies have shown microRNAs play very important roles in lipid metabolism in mammal, but much is unknown in fish. Our previous studies showed miR-122/23 involve in lipid metabolism in grass carp, several potential binding sites are found in 3’UTR of SREBP-1c. In this grant request, grass carp steatosis hepatocyte and in vivo model will be applied, overexpress and knockdown of miR-122/33 will be achieved by overexpression and interference vectors. Biochemical parameters, key genes’ expressions and enzyme activities of lipid metabolism will be determined by western blot, qRT-PCR and enzyme activity measurement; luciferase reporter system will be used to validate the potential binding sites of miR-122/33 in 3’UTR of SREBP-1c. Based on this, we will explore the role and molecular mechanism of miR-122/33 mediated by SREBP-1c in lipid metabolism in grass carp, and this will provide some key scientific directions for the prevention of fatty liver and other metabolic syndrome and for healthy growth of fish.

摘要：养殖鱼类普遍存在由于脂肪过度蓄积而导致的肝脂质代谢紊乱问题。以往研究发现miRNAs 通过作用于靶基因对脂肪的合成及分解等代谢生理过程起到重要调控作用。本项目通过高通量测序技术筛选出与肝脏脂肪代谢密切相关的miR-33/122, 构建了草鱼活体及细胞脂肪肝模型，并在细胞与活体水平上解析了miR-33/122调节鱼类肝脂代谢的过程以及分子机制。主要研究成果包括：（1）成功构建了高糖高脂饲料诱导的脂肪肝miRNA 文库，采用Hiseq深度测序技术，筛选出高糖高脂诱导下差异表达的miRNAs;（2）成功克隆了关键miRNA作用途径相关靶标基因SREBP-1的cDNA全长和3'UTR区; 并成功构建了SREBP-1 3'UTR荧光素酶报告基因载体，验证了SREBP-1是miR-33的候选靶基因。（3）采用细胞转染和腹腔注射miR-33/122的 mimic（tagonir）和agomir（antagonir）的方法，在细胞和活体水平上研究了miR-33/122的功能。结果表明，miR-33/122二者均可通过正向调节脂代谢和免疫相关基因等的表达在草鱼肝脂代谢紊乱过程中发挥重要作用，但二者的调节细节不同。（4）采用葡萄籽原花青素（GSPE）和草鱼肝细胞共孵育及草鱼腹腔注射GSPE的方法，发现GSPE能通过抑制miR-33和miR-122的表达进而调节一系列脂代谢和免疫相关基因有关。研究结果为丰富鱼类脂代谢调控机理提供了基础资料，为防治鱼类营养性脂肪肝等代谢性疾病及提高机体免疫力提供了思路及理论基础。