牛脂肪细胞增殖分化过程中关键候选lncRNA BADLNRs 的功能及其调控机制研究

Proliferation and differentiation of adipocyte is one of the most important factors for growth and development and meat quality in beef cattle. Its mechanism understanding will help us solve the problems of low growth rate and meat production in Chinese yellow cattle. Long non-coding RNAs (lncRNAs), which are not very conserved across species, play a significant role during the cattle preadipocyte proliferation and differentiation. However, we do not know the detailed crucial lncRNAs in adipocyte proliferation and differentiation, as well as their function and regulation mechanism are still unknown. Base on above facts, we intend to combine the existing data we have obtained from screening differentially expressed lncRNAs in progress of preadipocyte maturation, and we will identify four novel lncRNA BADLNRs (bovine adipocyte differentiation lncRNAs), namely, BADLNR1～4. Using overexpression and silence strategy, the function and regulation mechanisms of these candidate lncRNAs will be studied in adipocyte proliferation and differentiation, as well as their co-regulation mechanism with lncRNA-miRNA will be analyzed. In addition, the RIP and ChIRP techniques will be used to detect those proteins and chromatins interacting with BADLNRs, to elucidate the regulation mechanisms of lncRNA-miRNA-target protein process controlling the cattle adipocyte proliferation and differentiation. Briefly, this study aims to clarify functions and regulatory mechanisms of the key candidate lncRNAs in the preadipocyte proliferation and differentiation. These results will provide a solid theoretical foundation to improve beef cattle growth rate and meat quality.

脂肪细胞增殖分化是影响肉牛生长发育与肉质的重要因素之一，其机理研究有利于解决我国当前地方黄牛品种生长慢、产肉率低等问题。长链非编码RNA(lncRNAs)在物种间保守性低，对牛脂肪细胞增殖分化具有重要调控作用，但存在哪些关键lncRNAs及其功能、调控机制如何，尚不清楚。因此，本项目以秦川牛为对象，在已有前体脂肪细胞分化过程中差异表达lncRNA数据的基础上，鉴定出4个新lncRNA BADLNRs（即BADLNR1～4)，利用功能获得与缺失策略研究它们对脂肪细胞增殖分化的调控作用，探索lncRNA-miRNA的共调控机理；此外，借助RIP、ChIRP等技术研究与BADLNRs相互作用的蛋白和染色质，阐明关键lncRNA-miRNA-靶蛋白对牛脂肪细胞增殖分化的具体调控机制。该项目旨在阐明关键候选lncRNAs在脂肪增殖分化过程中的功能与调控机制，为肉牛快速生长与肉质改良奠定基础。

本项目以牛脂肪细胞为主要研究实验对象，探讨脂肪发育过程中关键候选lncRNA对脂肪发育的调控作用及功能机制。本项目在实验室前期牛脂肪细胞lncRNA测序结果中筛选了候选lncRNAs，同时结合秦川牛脂肪细胞的miRNA高通量测序结果，筛选并鉴定了脂肪发育过程中关键lncRNAs功能及其竞争性吸附miRNAs调控脂肪沉积的分子机制。本研究成功克隆了牛脂肪细胞增殖分化相关6个lncRNA 全长，并解析了其分子特征，并发现它们在物种间保守性低；利用功能获得与缺失策略研究了lncRNA BADLNR1、BADLNR2、BADLNR4、BADLNR5、lncCCPG1和lncSLC30A9等对脂肪细胞增殖分化和凋亡的调控作用，并解析了lncRNA BADLNR4-miR-204、lncCCPG1-miR-93、lncSLC30A9-miR-93共调控脂肪沉积的机理；借助ChIRP-Seq等技术发现了lncSLC30A9-FOS蛋白、lncCCPG1-FOS蛋白以及BADLNCR1-GLRX5等互作调控脂肪细胞的增殖分化。本研究阐明了关键lncRNAs-miRNA以及lncRNAs-miRNA-靶蛋白对牛脂肪细胞增殖分化的具体调控机制，为提高肉牛生长速度与肉质提供科学资料。此外，为推进脂肪增殖分化相关lncRNA及靶基因的育种应用，本研究还揭示BADLNR4、BADLNR5、miR-204和sirt1基因遗传变异与牛体重等性状显著相关，可作为选育的有效遗传标记，为肉牛快速生长与肉质改良奠定基础。基于以上研究成果，本项目已发表SCI论文16篇，其中IF>5.0的SCI论文4篇，中科院大类二区及以上SCI论文8篇，授权国家发明专利2项、获批软件著作权4项。