牛circMYBPC1调控成肌细胞增殖分化的分子机制解析

Proliferation and differentiation of myoblasts are important processes in muscle development, and dissecting the regulatory mechanism is necessary for the molecular breeding and meat quality improvement of livestock. Previously, we have identified a circMYBPC1 which expression in adult cattle muscle is significant higher than embryonic muscle, presumably circMYBPC1 plays an important role in the muscle development. however, the regulatory mechanism is unclear. Bioinformation analysis revealed that there is a miR-107 binding site in circMYBPC1sequence, and miR-107 inhibit the proliferation and differentiation of myoblast by targeting Wnt3a. Therefore, it is necessary to study the interaction between circMYBPC1 and miR-107. In this study, the transcription factor, RNA, and protein interacted with circMYBPC1were identified by using the techniques of ChIRP, RNA pull down and RIP. The objective is to investigative the transcription, function and mechanism of circMYBPC1, to uncover the regulatory relationship between circMYBPC1 and miR-107. Our results will comprehensively elucidate the regulation mechanism of circMYBPC1 on myoblasts proliferation and differentiation at the levels of transcription, post-transcription and signal transduction, and provide a new theoretical basis for molecular breeding at the levels of circRNA.

肌肉发育依赖于成肌细胞增殖和分化，研究肌细胞增殖分化的调控机制对家畜分子育种、提高肉品质具有重要科学意义。申请人鉴定到circMYBPC1在成年牛肌肉中表达水平远高于胚胎期肌肉中，推测其对肌肉发育有重要作用，但circMYBPC1调控作用机制尚不清楚。分析显示circMYBPC1序列中存在miR-107结合位点，而miR-107可通过调控Wnt3a表达抑制肌细胞增殖分化，故而有必要深入研究circMYBPC1与miR-107互作关系。本项目利用ChIRP、RNA pull down、RIP等试验技术鉴定circMYBPC1互作的转录因子、RNA、蛋白质，旨在解析circMYBPC1转录、功能及作用机制，阐明circMYBPC1与miR-107互作关系，从转录、转录后、信号转导等层次揭示circMYBPC1调控肌细胞增殖分化机制，在circRNA层面为肉牛分子育种提供依据和新思路。

背景：肌肉发育是影响家畜生长与经济效益的重要因素，直接影响肉质、风味、营养价值。然而，目前肌肉发育的分子机理研究基础还很薄弱，限制了肉牛分子育种的发展。肌细胞增殖分化是肌肉发育重要因素，研究肌细胞增殖分化的调控机制对家畜分子育种、提高肉品质具有重要科学意义。.研究内容：本项目首先对circMYBPC1的真实性及牛不同时期肌肉组织中表达水平进行鉴定；利用超表达及干扰等试验技术鉴定circMYBPC1对牛肌细胞增殖、分化的作用；利用双荧光素酶报告载体等手段阐明circMYBPC1与miR-107互作关系；筛选miR-107调控牛肌细胞增殖分化的下游靶基因，并探究了靶基因功能。.重要结果：本研究确定了circMYBPC1的真实性且在不同时期肌肉组中表达水平差异显著，利用重组载体pCD2.1-circMYBPC1及siRNA超表达及干扰circMYBPC1后，由CCK-8、EdU、RT-QPCR、western blot等试验结果显示：circMYBPC1促进牛肌细胞增殖、分化，circMYBPC1能吸附miR-107；miR-107可抑制肌细胞增殖、分化；miR-107可靶向作用FOXP1基因，抑制FOXP1 mRNA及蛋白水平表达；siRNA干扰FOXP1可抑制肌细胞增殖分化。.关键结果：circMYBPC1通过竞争性吸附miR-107调控基因FOXP1表达进而作用于肌细胞增殖分化。.科学意义：本研究通过揭示circMYBPC1调控肌细胞增殖分化的分子机制，有助于解析黄牛肌肉生长发育的分子机理，从circRNA层面为肉牛分子育种提供依据和新思路。