鸡Vanin-1基因的表达调控机理及其在肝脏脂类代谢中的功能研究

De novo synthesis of fatty acids in chickens occurs in liver. Vanin-1 is a pantetheinase that plays important roles in lipid metabolism in mammals, but its roles in lipid metabolism in chickens are still unknown. In this study, the RACE method, the bioinformatics, reporter gene assay, chromatin immunoprecipitation (ChIP) , electrophoretic mobility shift assay (EMSA), as well as site-directed mutagenesis will be combined to confirm the structure of Vanin-1 promoter, binding sites of transcription factors, and to understand regulatory mechanism of Vanin-1 gene in chickens. Through bioinformatics, miRNAs were screened which can directly bind to Vanin-1 gene 3’UTR. Then, the mechanism of post-transcription regulation of Vanin-1 gene was verified by co-transfection and dual-luciferase reporter assays. Using clustered regularly interspaced short palindromic repeat sequences interference (CRISPRi) system to knockdown the expression level of Vanin-1 in primary chicken hepatocytes, and differentially expressed genes in the chicken hepatocytes were detected by high throughput sequencing technology. Furthermore, detection of the expression of genes related to lipid metabolism was performed in chicken hepatocytes after over-expressing Vanin-1 by fluorescence quantitative PCR and Western Blot. Meanwhile the biochemical parameters related to lipid metabolism will be detected in the chicken hepatocytes with knockdown or over expression of Vanin-1 and in culture medium by biochemical methods. Taken together, the roles of Vanin-1 gene in chicken lipid metabolism will be concluded comprehensively. The results of this study will help to understand the molecular mechanism of lipid metabolism and control of fat deposition in chicken liver.

鸡脂类合成主要发生在肝脏中。Vanin-1是一种泛酰巯基乙胺酶，在哺乳动物中对肝脏脂类代谢具有重要的调控作用，而它在鸡脂类代谢中的功能尚不清楚。本项目拟采用RACE并结合生物信息学和荧光素酶报告基因的方法以及ChIP和EMSA、定点突变等技术，确定鸡Vanin-1基因的启动子结构、转录因子精确结合位点，弄清该基因的转录调控机制；通过生物信息学筛选出能与该基因配对结合的miRNA，并用双荧光素酶报告基因进行验证以研究其转录后调控机制；采用过表达和CRISPRI基因干扰技术，分别在鸡LMH细胞系和原代鸡肝细胞中过表达和敲低该基因，高通量测序检测肝细胞中差异表达基因，并检测细胞及培养基中与脂质代谢相关的生化指标，运用qRT-PCR和Western Blot检测脂质代谢相关基因的表达量，综合分析Vanin-1在鸡肝脏脂类代谢中的功能。研究结果将有助于认识鸡肝脏脂类代谢和控制脂肪沉积的分子机理。

VNN1是一种泛酰巯基乙胺酶，在哺乳动物肝脏的糖脂代谢中发挥重要的作用。以弄清鸡VNN1基因表达调控分子机理及其在肝脏脂类代谢中的功能为目标。确定了鸡VNN1基因的转录起始位点位于起始密码ATG上游42bp处，其核心启动子区在-1至-589bp；发现该基因启动子区具有转录因子C/EBPβ、HNF4α和PPARα结合位点。C/EBPβ、HNF4α和PPARα结合位点突变后显著降低报告基因转录活性，LMH细胞中过表达或抑制HNF4α能显著影响该基因表达，抑制PPARα和PPARα激动剂显著影响该基因的表达。表明VNN1基因表达受HNF4α、PPARα和C/EBPβ的正调控。运用miRanda预测到251种靶向作用于鸡VNN1基因的miRNA，证明miR-33-5p和miR-181a-5p调控鸡VNN1基因表达。用RNAi抑制原代鸡肝细胞VNN1的表达，高通量测序分析发现共有376个DGE，其中138个上调，238个下调；GO分析表明25个DEG参与鸡肝细胞脂类代谢过程，KEGG分析显示DEG在16个脂类代谢途径中显著富集；发现VNN1抑制后FASN、ELOVL2和GDPD5的表达量显著上升，E2F7、WNT5B、PLA2G12A和RACGAP1的表达量显著下降。用CRISPR/Cas9基因编辑将LMH细胞VNN1敲除后进行RNA-seq分析，发现有1335个DEG，其中431个上调，904个下调；GO分析表明脂质代谢途径如调节脂肪酸生物合成与代谢、长链脂肪酸生物合成过程等被富集；KEGG分析表明泛酸和CoA生物合成、甘油脂代谢、不饱和脂肪酸的生物合成、脂肪酸生物合成等途径被富集；发现76个脂质代谢相关DEG，其中29个上调，下调47个，表明VNN1在鸡肝脂代谢过程中起着重要作用。研究结果将有助于认识鸡肝脏脂类代谢和脂肪沉积的分子调控机理，为提高鸡的肉质和生产效率提供新的思路。