鸡SIRT1基因的表达调控及其对肝脏脂类代谢的功能研究

Sirtuin1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that regulates lipid metabolism in mammalian. De novo fatty acid synthesis in chickens takes place mainly in the liver, whereas the role of SIRT1 gene in lipid metabolism in chickens has not been reported. In this study, the RACE method will be used to obtain full length cDNA sequence of SIRT1 gene, and the bioinformatics, reporter gene assay, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) will be combined to understand the structure of SIRT1 promoter and regulatory mechanism of SIRT1 gene in chickens. Through bioinformatics, miRNAs were screened which can directly bind to SIRT1 3'UTR. Then, the mechanism of post-transcription regulation of SIRT1 gene was verified by co-transfection and dual-luciferase reporter assays. Using siRNA to knockdown the expression level of SIRT1 in chicken hepatocytes, and differentially expressed genes in the chicken hepatocytes were detected by RNA-Seq technology in the meantime. Furthermore, detection of the expression of genes in signaling pathways was performed in chicken hepatocytes after over-expressing SIRT1 by fluorescence quantitative PCR and Western Blotting. Accordingly, the role of SIRT1 gene in chicken lipid metabolism will be concluded comprehensively. The regulatory mechanism of lipid metabolism and fat deposition of chicken will be explored from another angle in this study.

SIRT1是一种NAD+-依赖性蛋白去乙酰化酶，哺乳动物中SIRT1基因对脂类代谢具有调节作用。鸡脂类合成主要在肝脏中完成，而SIRT1基因在鸡脂类代谢中的功能尚未报道。本研究将采用RACE方法，获得鸡SIRT1基因全长cDNA，结合生物信息学方法、报告基因分析手段、ChIP和EMSA等技术，弄清鸡SIRT1基因启动子结构和该基因的转录调控机制；通过生物信息学的方法，筛选出能与SIRT1基因配对结合的miRNA，并用双荧光素酶报告基因进行细胞学验证以研究其转录后调控的机制；同时利用转染siRNA的方法，敲低鸡肝细胞中SIRT1的表达，结合RNA-Seq技术检测肝细胞中差异表达基因；在肝细胞中过表达SIRT1，通过荧光定量PCR和Western Blotting方法检测SIRT1信号通路中基因表达情况，综合分析SIRT1基因在鸡脂类代谢中的作用，从另一角度探讨鸡脂类代谢和脂肪沉积的调控机理。

SIRT1联系着机体的代谢水平和基因转录的网路调控。为揭示SIRT1在肝脏这一鸡重要的脂类代谢器官中的作用和功能，确定了以研究鸡SIRT1基因表达的转录调控和miRNA对其转录后的调控机制，以及其对鸡肝脏脂类代谢的调控机制为研究目标。通过5′RACE技术扩增获得了鸡SIRT1基因14 bp长的5′UTR序列，经生物信息学方法分析和序列删除实验预测并证实了影响该基因转录活性的关键区域位于转录起始位点上游-114~-98之间，在-151~-116之间存在对SIRT1基因转录具有促进作用的元件。预测到该基因的第一个外显子及其上游序列中存在一个约1600 bp长的CpG岛，5-氮杂胞嘧啶核苷抑制LMH细胞的甲基化实验结果表明甲基化可抑制鸡SIRT1基因的表达。预测发现136个潜在作用于鸡SIRT1基因的miRNAs，证实gga-miR-204在mRNA和蛋白水平可抑制鸡肝脏细胞中SIRT1基因的表达。利用siRNA干扰鸡原代肝脏细胞中SIRT1基因的表达后，RNA-seq分析发现鸡肝细胞中有86个基因发生了差异性表达，其中63个基因表达下降，23个基因表达上升。差异表达基因Adh6、H6pd、Hexa_b和glmS与碳水化合物代谢有关，Adh6、Acsl4、Lipa、Gnpat与脂类代谢有关，Vnn1、Mt3（表达减少）和Frzb（表达增加）分别与肝脏的糖脂类代谢和肝细胞的发育有关。SIRT1的瞬时过表达证实了该基因对LMH细胞中Lipa、Frzb、Adh6、Acsl4、Vnn1和Mt3的表达调控。在构建的稳定敲低SIRT1的LMH-gSmiR30细胞系中，SIRT1基因的低表达改变了H6pd、G6pc和Vnn1基因在LMH细胞中的表达，影响了LMH细胞对葡萄糖应答反应，同时降低了LMH细胞中G6pc、Atgl对高糖高脂的应答，抑制了糖酵解和脂肪的降解，促使细胞内脂肪滴的形成。