miR-122在吉富罗非鱼肝脏脂肪沉积中的作用及其调控机制研究

Liver is an important organ for lipid metabolism regulation. The tilapia fed high lipid diets for a long term is subjected to lipid deposition in liver, which can affect growth, meat quality and disease resistance. miRNA has been proven to be crucial regulation factor and involved in a variety of signal transduction. Up to now, the regulation mechanism of miRNA related to lipid deposition in tilapia remains unclear. The comparative results of miRNA expression profiling between the tilapia fed normal diets and high lipid diets showed that miR-122 is closely related to lipid deposition. In this study, by taking miR-122 as candidate, we attempt to build evaluation system of stress in tilapia fed high lipid diets firstly; the potential target gene of miRNA will be verified by bioinformatics analysis, the luciferase reporter gene system and electrophoretic mobility shift assay(EMSA); the expression characteristics of miRNA and its target gene will be analyzed through real time polymerase chain reaction (qRT-PCR), Northern blot and Western blot; the protein distribution in tissues and in hepatocytes will be studied through immunohistochemistry (IHC) and immunofluorescence (IF) technology; potential signal transduction pathways will be deeply investigated by over-expression and locked nucleic acid (LNA)-expression of miRNA at the cell and overall levels. The results of this project will provide useful clues for the miRNA regulation on the mechanisms of lipid deposition in tilapia liver. Also, the project will benefit the investigation of regulation pathway, and adding new and valuable academic information for solving the disease in lipid metabolism.

肝脏是重要的脂代谢调控器官，长期摄食高脂肪饲料易引发罗非鱼肝脏脂肪沉积，影响生长、肉质与抗病力。miRNA是一种重要的基因调控分子，参与了多种信号传导过程。关于miRNA调控罗非鱼脂肪沉积的分子机制尚未清楚。前期通过miRNA表达谱差异分析，发现miR-122与罗非鱼肝脏脂肪沉积密切相关。本项目拟以miR-122为研究目标，首先构建高脂应激评价体系，通过生物信息学、荧光素酶报告基因系统与凝胶阻滞技术确定miRNA的上游调控基因；采用qRT-PCR、Northern blot和Western blot分析miRNA与靶基因表达特征；采用免疫组化和免疫荧光技术研究靶基因的组织定位与细胞定位；通过过表达与抑制目标miRNA从细胞与整体水平深入分析潜在的信号调控通路。研究结果将为揭示miRNA调控罗非鱼肝脏脂肪沉积的分子机制提供有益线索，为研究脂代谢相关调控途径，解决脂代谢类疾病提供新的学术资料。

肝脏是重要的脂代谢调控器官，长期摄食高脂肪饲料易引发罗非鱼肝脏脂肪沉积，影响生长、肉质与抗病力。miRNA是一种重要的基因调控分子，参与了多种信号传导过程。本项目中，我们首先构建了高脂应激模型。高脂饲喂（18.5%）会导致过量的脂质在吉富罗非鱼肝脏中沉积，并对吉富罗非鱼生长，血清生化指标，肝脏脂肪酸组成，肝脏组织结构，抗氧化能力造成不利影响。利用Solexa HiSeq 2000测序仪建立了高脂应激组（18.5%）与对照组（8%）肝脏miRNA转录组文库，分析了与脂代谢相关miRNA的变化及其潜在的靶基因。我们利用生物信息学与双荧光素酶法验证了硬脂酰辅酶 A 去饱和酶（SCD）是miR-122的靶基因。同时，我们将带有miR-122的目的质粒转染罗非鱼肝细胞，48h和72h时，添加50nM或100nM miR-122 mimic的共转染实验组细胞中荧光素酶活性显著低于对照组；miR-122 mimic组48h和72h时，SCD的蛋白表达量也明显减少，表明miR-122与其靶基因SCD呈负相关调控。同时，qRT-PCR分析显示，50nM或100nM miR-122 mimic组SCD mRNA表达水平也显著低于对照组。说明上调或下调miR-122表达可以反向调控SCD基因的表达。高脂组miR-122表达水平在高脂应激后第20d和第40d时显著高于（P<0.05）对照组。但是，第60d时高脂组miR-122表达水平出现下降，并与对照组相比差异不显著。这与northern blot结果一致。吉富罗非鱼肝脏SCD对高脂应激后急剧下降，高脂应激后第20d, 40d和60d，高脂组的SCD转录水平与蛋白水平均显著低于对照组。通过尾静脉注射miR-122 antagomir研究吉富罗非鱼体内miR-122的调节通路。抑制miR-122表达可以上调SCD基因表达，进而干扰脂代谢调控过程。高脂应激时，肝脏中上调的SCD基因有助于促进脂肪合成基因（SREBP-1和LPL）的表达，增加肝脏TC与TG含量，从而导致吉富罗非鱼肝脏脂肪沉积的发生。本项目的实施弥补了miRNA对鱼类脂肪沉积的调控研究的空白。同时，本项目的开展对于了解罗非鱼脂肪沉积的分子机制，以及罗非鱼营养性疾病的治疗，提高罗非鱼肝脏应激保护有着重要的生物学意义。