环状RNA circMDs调控牛成肌细胞增殖分化的机制研究

Proliferation and differentiation of myoblasts are important processes that affect growth, development and meat production of beef cattle. Studying the mechanisms will be of benefit to the growth speed and meat production rate of Chinese beef cattle. Recently, research on circRNAs has become a hot issue and many studies indicated that circRNAs play crucial roles in proliferation and apoptosis of cancer cells. However, the regulatory roles of circRNA in bovine myoblasts proliferation and differentiation remain unknown. Therefore, based on circRNA sequencing data in longissimus dorsi muscle of Qinchuan and Angus cattle, we intend to identify and verify the differentially expressed circRNAs in two developmental stages. Moreover, we will perform expression pattern analysis, over expression, and RNA interference methods to explore the vital functions of circRNAs in proliferation and differentiation of bovine myoblasts. Thereafter, the functional circRNAs will be named as circMDs. After that, the technologies of Subcellular localization, Dual luciferase reporter system, qRT-PCR, Western blot, ChIRP, RIP and in vivo animal model will be utilized to uncover the molecular mechanism underlying circMDs regulating the bovine myoblasts proliferation and differentiation. In summary, our study will provide basic theoretical foundations for cultivating new breeds of Chinese beef cattle.

成肌细胞增殖分化是影响肉牛生长发育和产肉量的重要因素之一，研究其机理有利于解决中国黄牛品种生长慢、产肉率低等问题。环状circRNA已成为一个新的热点研究领域。研究表明，circRNA对癌细胞的增殖、凋亡有重要调控作用，但对牛成肌细胞增殖分化是否具有调控作用及其调控机制尚不清楚。因此，本课题组以秦川牛背最长肌circRNA测序结果为基础，以安格斯牛为对照，筛选并鉴定出不同品种、不同发育时期差异表达的circRNAs；通过组织表达分析、超表达和干扰等方法，研究circRNA对牛成肌细胞增殖分化的影响，以确定对牛肌肉发育具有重要功能的circMDs。同时通过circMDs的亚细胞定位、双荧光素酶报告基因系统、qRT-PCR、Western blot、ChIRP、RIP等技术和动物模型活体验证，系统揭示circMDs调控牛成肌细胞增殖分化的分子机制，为培育优良中国肉牛新品种提供理论依据。

本课题以秦川牛肌肉作为主要研究实验对象，利用去除核糖体RNA高通量测序技术对不同发育时期的肌肉组织circRNA进行筛选与鉴定，同时运用生物信息学和多种分子生物学手段挖掘对牛肌肉发育具有重要功能的circRNA并系统揭示其调控的分子机制。通过对胚胎期90天和24月龄成年牛背部肌肉组织circRNA的差异比较分析，得到了828个差异表达的circRNAs。我们选取了20个表达差异的circRNAs，利用反向引物扩增、RNase R处理、放线菌素D处理等实验验证circRNA的真实性与稳定性；利用超表达与干扰技术，研究这些circRNA对牛肌肉原代细胞发育的影响；接下来利用RNA FISH、双荧光素酶检测系统、RNA pull down、RIP等试验研究定位于细胞质和细胞核中的circRNA调控牛肌肉细胞发育的分子机制；最后通过构建小鼠肌损伤模型验证circRNAs在体内对肌肉发育的功能。此外，通过生物信息学分析处理构建了circRNA-miRNA-mRNA三元共表达网络，用以预测秦川牛骨骼肌发育过程中circRNA介导的潜在ceRNA网络。本项目还对秦川牛成年牛以及犊牛脂肪组织circRNA进行了测序分析，并研究了circRNA调控牛脂肪细胞发育的功能及其机制。作为参考，还增加了lncRNA和miRNA调控牛肌肉和脂肪细胞发育的功能与机制研究。最后本项目还分析了牛脂肪细胞释放的外泌体的RNA转录组，从而扩展了我们对脂肪细胞外泌体RNA的生理功能的理解。因此，本项目从非编码RNA层面破解秦川牛肌肉生长和肉质性状形成的遗传机制，从而对提高中国黄牛的生长速度和肉品质等方面具有极其重要意义。本项目共在Science China Life Science、Journal of Animal Science and Biotechnology、Molecular Therapy - Nucleic Acids、Cell Death and Disease等杂志发表SCI论文28篇，获2件授权国家发明专利。已按计划很好的完成了项目的研究任务。