高脂诱导肥胖型五指山猪肝脂代谢相关lncRNA的鉴定及其调控机制研究

At present, most attention is focused on obesity because it is associated with an increased risk of developing type 2 diabetes , metabolic disorder and cancer. In fact, the recent dramatic increase in knowledge has demonstrated that long noncoding RNA (lncRNAs) are involved in adipogenic gene regulatory network, but the precise molecular mechanism about how lncRNAs regulated liver lipid metabolism caused by obesity has largely remained elusive. Because the minipig is the ideal animal model of human obesity and metabolic disease, in the present study, we will performe Ribo-Zero RNA-Seq to illuminate different expression of lincRNA in the liver tissue with obese wuzhishan pig model induced by high fat diets. In addition, we explored the regulatory mechanism of lncRNA on the liver cell differentiation by using some latest technology including RACE, dual luciferase reporter assay and WB to preliminarily reveal the regulate molecular mechanism of liver fat differentiation on lncRNAs as ceRNA through interaction with combination of microRNAs. The predicted results of this project will be helpful to provide the theory basis for the molecular mechanism of liver lipid metabolism related to high-fat diet nutritional obesity.

随着经济的发展和生活水平的提高，我国肥胖人数也在快速增多，由肥胖引发的糖尿病、代谢紊乱症及癌症等的发病率也逐年升高，严重威胁到人类健康，使得肥胖问题成为当今人们关注的焦点。近年来的研究显示长链非编码RNA对于脂肪生成具有重要的调控作用，但目前关于 lncRNAs 对肥胖引起的肝脂代谢相关的研究还未见报道。由于小型猪是研究人类肥胖及代谢相关疾病的理想动物模型，因此，本研究以高脂诱导的五指山猪肥胖模型为研究对象，采用高通量测序技术分析在不同肝脏组织表达的 lncRNA，筛选可能参与肝脏脂肪代谢的 lncRNA；并利用 cDNA末端快速扩增、基因过表达、双荧光素酶报告系统和Western Blot等最新技术初步揭示lncRNAs 作为ceRNA 通过与结合miRNA相互作用调控肝脏脂肪分化的分子机制，为解析与高脂膳食密切相关的营养性肥胖引起的肝脂代谢的分子机制提供理论依据。

由肥胖引发的糖尿病、代谢紊乱症及癌症等问题成为当今人们关注的焦点。近年来的研究显示长链非编码RNA对于脂肪生成具有重要的调控作用，但目前关于 lncRNAs 对肥胖引起的肝脂代谢相关的研究还未见报道。小型猪是研究人类肥胖及代谢相关疾病的理想动物模型，本项目利用高脂饲喂的方法建立了五指山猪肥胖模型，通过高通量测序的方法筛选了差异的lncRNA，发现lncRNA LOC106509199在差异的lncRNA中表达量最高，因此我们以lncRNA LOC106509199（HLMNCR）为候选基因，与miRNAome 数据整合分析后发现HLMNCR序列上存在有 miR-708-5p 的结合位点，荧光素酶活性分析试验表明miR-708-5p能够靶向HLMNCR，软件分析和文献查阅后聚焦到miR-708-5p的靶基因GPAM。荧光素酶活性分析试验表明miR-708-5p能够靶向GPAM。猪原代肝细胞中过表达ps-miR-708-5p载体后的油红O染色结果显示，相同转染效率的情况，过表达miR-708-5p的细胞中脂滴数量减少，细胞中甘油三酯含量极显著降低，miR-708-5p 能显著降低GPAM蛋白的表达水平。同样，过表达psHLMNCR 能显著增加肝细胞中甘油三酯含量以及GPAM蛋白的表达量，而过表达HLMNCR 对GPAM 的突变体却不能改变。双荧光素酶报告系统竞争性结合试验也证明HLMNCR 可以与GPAM 竞争性结合miR-708-5p。以上结果证实HLMNCR通过 lncRNA HLMNCR →miR-708-5p→GAPM网络调控肝脏脂肪合成。