蛋鸡miR-223表达调控的分子机制及其在肝脏脂质代谢中的功能研究

De novo synthesis of fatty acids in chickens occurs in liver. Fatty liver and lipid deposition in yolk in laying hens are related with lipid metabolism in liver. miR-223 is an important miRNA that plays important roles in lipid metabolism in mammals, but the roles of it in lipid metabolism in chickens are still unknown. In this study, RT-qPCR will be used to detect the temporal and spatial expression of miR-223 in various tissues, in livers of various stages in laying hens. 5'RACE, bioinformatics, luciferase reporter gene assay, chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) and site-directed mutagenesis will be combined to identify the binding sites of transcription factors in the promoter of miR-223 gene. Through over-expression and RNA-interference of transcription factors, we will analyze the regulatory mechanism of miR-223 expression. Bioinformatics will be used to predict the target genes of miR-223, in which the genes related to lipid metabolism will be chosen and dual-luciferase reporter system and site-directed mutagenesis will be used to validate them. In chicken hepatocytes, miR-223 will be over-expressed and knocked down, and the biochemical index related to lipid metabolism will be detected by biochemical methods and the expression levels of target genes and genes related to lipid metabolism in chicken liver will be detected by RT-qPCR and Western Blot to analyze the functions of miR-223 in lipid metabolism in laying hen liver. The results of this study will help to understand the molecular mechanism of lipid metabolism in laying hen liver, and provide the theoretical basis and new idea for the improvement of fatty liver and yolk quality in laying hens.

鸡脂类合成发生在肝脏中，蛋鸡脂肪肝和卵黄脂质沉积与肝脏脂质代谢密切相关，miR-223是一种重要的miRNA，在哺乳动物中对肝脏脂质代谢具有重要调控作用，而它在蛋鸡脂质代谢中的功能尚不清楚。本研究将采用RT-qPCR检测miR-223在蛋鸡不同组织、不同时期肝脏中时空表达特征；结合生物信息学、启动子报告基因和ChIP-qPCR等鉴定miR-223基因启动子转录因子结合位点；过表达和RNA干扰转录因子，分析miR-223表达调控机制；采用生物信息学方法预测miR-223靶基因，挑选其中与脂质代谢相关基因，用双荧光素酶报告系统和定点突变进行验证；分别在鸡肝细胞中过表达及敲低miR-223，用生物化学法检测脂质代谢相关生化指标，并进一步检测相关基因的表达，分析miR-223在蛋鸡肝脏脂质代谢中的作用。研究结果将有助于认识蛋鸡肝脏脂质代谢的分子机理，为改善蛋鸡脂肪肝和蛋黄品质提供理论基础和新思路。

miR-223是脂质代谢中重要的miRNA，可能与蛋鸡脂肪肝有关，鸡miR-223的表达及其在肝脏脂质代谢中的功能有待进一步研究。本研究运用RT-qPCR检测蛋鸡miR-223的时空表达，发现其在肺组织高表达，肾组织低表达，脂肪组织、肝脏等组织中等表达，在16~56周龄肝脏中的表达呈下降趋势，与腹脂率相反。通过生物信息学预测到转录因子HNF4A与miR-223基因启动子结合，通过双荧光素酶报告实验、ChIP-qPCR和定点诱变技术证实了结合位点。鸡肝细胞中过表达和RNA干扰HNF4A，证实HNF4A能与miR-223基因启动子结合，上调miR-223的表达。运用靶基因预测软件预测到miR-223的靶基因DAGLA，通过双荧光素酶报告实验证实了DAGLA是miR-223的靶基因，靶位点突变实验也证实了预测的靶位点。鸡肝细胞中过表达和敲低miR-223，证实了miR-223对靶基因DAGLA和其他脂质代谢相关基因FASN、ACACA在mRNA水平和蛋白水平均具有负调控作用，且通过油红O检测和TG、TC、FFA等生化指标检测发现miR-223能抑制鸡肝细胞脂质沉积，促进鸡肝细胞脂质分解。研究结果有助于对调控肝脏脂质代谢，从而改善蛋鸡脂肪肝提出新的策略。