linc_000677与EBF1的互作及其在牛胎儿骨骼肌MSCs成脂分化中的调控机制研究

The intramuscular adipocytes are derived from the mesenchymal stem cells (MSCs) in fetal bovine skeletal muscle.There is a competition between adipogenesis and myogenesis,fibrogenesis.Therefore,modulating fetal bovine skeletal muscle MSCs adipogenesis differentiation is a potential way to increase intramuscular fat.It is suggested that lncRNAs is an important regulatory molecule during MSCs differentiation to adipocytes.Unfortunately, our knowledge of how fetal bovine skeletal muscle MSCs adipogenesis is under the lncRNAs control is limited.Previously,an up-regulated lncRNA named linc_000677 that was identified before and after adipocyte differentiation of fetal bovine skeletal muscle MSCs by sequencing, the genomic loci of linc_000677 locates at upstream of EBF1,which a transcription factor of regulating adipocyte differentiation,and both linc_000677 and EBF1 have similar expression patterns.Therefore in this research,using fetal bovine skeletal muscle MSCs adipogenesis as experiment model,the relationship between linc_000677and EBF1,and also the cell signaling pathway they were involved,were investigated using technologies including RNA interference,ChIP,dual luciferase report assay.Finally, the regulation mechanism of linc_000677 in fetal bovine skeletal muscle MSCs differentiation to adipocytes will be characterized.It will provide the theory basis to investigate molecular mechanism of intramuscular fat deposit in beef cattle.

牛肌内脂肪细胞是由胎儿骨骼肌间充质干细胞（MSCs）分化而来，其与肌细胞和成纤维细胞的形成存在竞争关系，因此调节胎儿骨骼肌MSCs成脂分化对肌内脂肪的形成具有重要意义。研究表明lncRNAs是控制MSCs成脂分化的重要调控分子，然而有关牛胎儿骨骼肌MSCs成脂分化相关的lncRNAs的研究至今未见报道。先前通过测序手段发现了一个在牛胎儿骨骼肌MSCs成脂分化前后表达上调的lncRNAlinc_000677，其基因位于脂肪生成相关转录因子EBF1上游，且二者表达模式相近。因此本研究以胎牛骨骼肌MSCs成脂分化为模型，利用RNA干扰、ChIP、荧光素酶报告基因等技术，探讨linc_000677与EBF1之间的调控关系及参与的细胞信号通路，揭示linc_000677在牛胎儿骨骼肌MSCs向脂肪分化过程中的调控机理，为进一步阐明牛肌内脂肪沉积的分子机制提供理论依据。

牛肌内脂肪细胞是由胎儿骨骼肌间充质干细胞（MSCs）分化而来，其与肌细胞和成纤维细胞的形成存在竞争关系，因此调节胎儿骨骼肌MSCs成脂分化对肌内脂肪的形成具有重要意义。本研究以linc_000677和EBF1为研究对象，开展linc_000677全序列的扩增、核质定位、时空表达模式的研究，利用siRNA探讨EBF1对其表达的影响，同时开展牛胎儿骨骼肌MSCs成脂分化体系的优化及转录组学研究。结果获得linc_000677 5′和3′端基因序列，linc_000677在核、质中均有表达分布，EBF1与linc_000677在牛胎儿骨骼肌MSCs成脂分化后2d表达水平最高，EBF1干扰在诱导分化初期不能改变linc_000677表达水平；VC和 FGF2 能明显改善牛肌源干细胞成脂能力，不同代次牛胎儿骨骼肌MSCs成脂分化转录组差异明细，代次越低分化潜能越高，Lin7A、MYF5、PTPRQ及SCL29A1可作为牛肌源干细胞成脂潜能的标记分子。该项目的实施将揭示linc_000677在牛胎儿骨骼肌MSCs向脂肪分化过程中的调控机理，为进一步阐明牛肌内脂肪沉积的分子机制提供理论依据。