绵羊胚胎骨骼肌发育相关环状RNA的筛选和功能研究

Circular RNA (circRNA) in animals are an enigmatic class of RNA with unknown function, and play an important role in organ development, cell proliferation and differentiation, and disease etc. However, the expression and function of circRNA in sheep skeletal muscle is not clear yet. We will choose Suffolk sheep and Kazak sheep with obvious difference of meat performance as research object. circRNA expression will be analyzed in skeletal muscle of sheep embryo using high-throughput sequencing and bioinformatics analysis. The significantly differentially expressed circRNA will be filtered; The interactions between circRNA and target gene will be identified by using luciferase gene reporting system. Expression and biological function of circRNA in the process of muscle cell proliferation and differentiation will be analyzed by overexpression and gene silencing technology. Our study can further enrich the circRNA database of mammals, and provides the basis for research on development-related circRNA of skeletal muscle. CircRNA can be used as potential molecular markers and functional gene for application in breeding of the mutton sheep.

环状RNA（circular RNA，circRNA）是新发现的一类特殊的非编码RNA，在动物细胞增殖分化、器官发育、疾病发生等过程中发挥重要作用。然而，circRNA在绵羊骨骼肌中的表达和功能迄今尚不清楚。本项目将以产肉性能明显差异的萨福克羊和哈萨克羊为研究对象，利用高通量测序技术分别构建两个绵羊品种的胚胎期骨骼肌circRNA表达谱，经生物信息学和定量分析筛选出品种间显著差异表达的circRNA；利用荧光素酶基因报告系统分析circRNA与靶基因的互作；利用circRNA过表达和基因沉默技术研究circRNA在成肌细胞增殖和分化过程中的生物学功能。本研究可进一步丰富哺乳动物circRNA数据库，并为动物骨骼肌发育相关circRNA的生物学功能研究提供依据。circRNA可作为分子标记和候选功能基因应用于我国肉羊新品种培育。

环状RNA（circular RNA，circRNA）是新发现的一类特殊的非编码RNA，在动物细胞增殖分化、器官发育、疾病发生等过程中发挥重要作用。本项目从RNA-seq数据中检测到6113个环状RNA，随机检测10个均是环状RNA，经验证环状RNA对RNase R具有抗性。生物信息学分析发现一些环状RNA可能参与调控肌肉生长和发育如circRNA-HIPK3。circRNA-HIPK3在小鼠肾组织中表达量最高，在骨骼肌中表达量较高。circRNA-HIPK3在C2C12细胞增殖与分化过程中，表达量均上调；干扰circRNA-HIPK3后，分化相关基因MyoG和MyHC表达量下调，并且C2C12细胞增殖率降低。通过生物信息学成功预测到circRNA-HIPK3可能吸附miR-30a-3p、miR-124-5p、miR-379-5p；构建的这些microRNA的过表达载体均可以高效表达；通过双荧光素酶试验最终确定circRNA-HIPK3可以吸附miR-124-5p 和miR-379-5p。本项目通过RNA-seq分析获得大量的环状RNA，肌肉中的环状RNA的表达谱为探究非编码RNA在肌肉生长发育中的作用提供了重要参考。通过生物信息学和分子生物学手段证明circRNA-HIPK3抑制C2C12成肌细胞增殖、促进C2C12细胞分化，对肌肉生长发育具有重要的影响。本项目阐明了circRNA-HIPK3调控肌肉生长发育的分子机制，为调控circRNA促进绵羊骨骼肌生长发育提供了理论支持。