miR-23a/27a/24-2簇调控牛肌内前体脂肪细胞增殖分化的协作机制

Intramuscular fat (IMF) or marbling positively affects meat palatability such as tenderness, juiciness. Studies on the mice have confirmed that miR-27a regulated adipogenesis by targeting PPARγ gene. However, the effects of miR-23a/27a/24-2 cluster on adipogenesis are not yet reported. Preview research found that the gene sequences of this miRNA cluster are highly conserved between bovine and other species, this cluster might regulate adipogenesis by influencing the expression of PPARγ directly or indirectly. But the function of miR-23a/27a/24-2 cluster and its cooperation mechanism are still unclear. The program is about to figure out the effects of miR-23a/27a/24-2 cluster on bovine intramuscular preadipocyte proliferation and differentiation by the gene over expression and RNAi methods, then explore the downstream targets of miR-23a/27a/24-2 cluster. Meantime, analyze the role of this miRNA cluster on downstream signaling pathways. This study can not only illuminate the cooperation mechanism of miR-23a/27a/24-2 cluster in regulating proliferation and differentiation of bovine intramuscular predipocytes, as well as help us to better understand molecular regulation network in intramuscular fat deposition, but also provide a theoretical basis for molecular breeding of beef cattle.

肌内脂肪含量是影响牛肉品质的重要因素之一。小鼠上已证实miR-27a靶向PPARγ调控前脂肪细胞成脂分化，但miR-27a所在的miR-23a/27a/24-2簇在脂肪生成中的协作效应机制尚未见报道。前期工作初步表明，该簇基因序列在牛及其它物种之间高度保守，它的3个成员可能通过直接或间接的方式影响PPARγ表达，相互协作参与调控牛肌内脂肪代谢，其分子机制有待进一步阐明。因此，本项目拟采用基因过表达、沉默技术，明确 miR-23a/27a/24-2簇在牛肌内前体脂肪细胞增殖分化中的具体作用；利用miRNA报告基因系统，鉴定其下游靶基因，同时探讨该miRNA簇对下游信号通路的调控机制。研究结果将阐明miR-23a/27a/24-2簇调控牛肌内脂肪细胞增殖分化的协作机制，为肌内脂肪沉积的分子调控网络解析和肉牛分子育种提供理论依据。

肌肉脂肪含量是影响牛肉品质的重要因素之一。研究表明miR-23a~27a~24-2簇在细胞代谢过程中起到非常重要的作用，但该miRNA簇在牛前体脂肪细胞分化中的作用及其协同作用机制尚不明确。本课题分离培养了新生秦川牛前体脂肪细胞，利用miRNA agomir和antagomir介导miR-23a~27a~24-2簇在前体脂肪细胞中过表达和干扰，观察miR-23a~27a~24-2簇对脂肪细胞分化的作用并探究了其下游靶基因及作用通路。本课题研究表明，通过胶原酶消化法，我们分离得到了具备良好生长状态和分化性能的秦川牛前体脂肪细胞。miR-23a~27a~24-2簇的表达随脂肪细胞的分化而升高，并且该miRNA簇在脂肪细胞分化过程中起抑制作用。miR-23a~27a~24-2簇对脂肪细胞分化的抑制作用是通过靶向两类基因来实现的：miR-27a和miR-24-2靶向GPAM和DGAT2，这两个基因干扰后抑制脂肪细胞分化；miR-23a和miR-24-2靶向DCN，G6PD和LPL，这三个基因干扰后促进脂肪细胞分化。通过对miR-23a~27a~24-2簇靶基因的进一步预测筛选，我们发现miR-23a，miR-27a和miR-24-2可以共同靶向调控FGF11的表达来抑制脂肪细胞分化。综上，本课题研究表明miR-23a~27a~24-2簇可同时靶向促脂生成靶基因和抑脂生成靶基因来平衡调控前体脂肪细胞的成脂分化。本研究不仅为脂肪发育研究提供了一定的研究基础，而且为秦川牛分子育种和基因组选择育种提供了理论依据。