牛前体脂肪细胞增殖分化过程中关键环状RNA circADs的鉴定及调控机制研究

Proliferation and differentiation of adipocyte is one of the most important factors for deposition of fat and meat quality in beef cattle. Thus uncovering the mechanism of adipocyte proliferation and differentiation is the foundation of promoting the development of Chinese cattle industry. CircRNA is a class of non-coding RNAs with important functions. It participates in the regulation of various biological processes, but its function in regulating bovine preadipocyte proliferation and differentiation has not been reported yet. Therefore, the objective of this study will screen the differently expressed circRNAs between preadipocyte and adipocyte of Qinchuan cattle basing on the high-throughput sequencing and bioinformatic analysis, as well as to choose 4~6 key circRNAs circADs via overexpression and interference strategies. Furthermore, the nuclear and cytoplasm separation techniques will be used for circADs subcellular localization. Flow cytometer and other techniques will be used to uncover the regulation of proliferation and differentiation of circADs for cattle preadipocyte, as well as the interaction among circADs, miRNAs and miRNAs’ target genes will also be built. Finally, the function of circADs in vivo will be verified by injecting circADs to mice inguinal fat and detecting the proliferation and differentiation of adipocyte. As a result, this study will illuminate the regulation mechanism of key circADs on adipocyte proliferation and differentiation and provide theoretical foundation for improving and breeding excellent beef cattle breed.

脂肪细胞增殖分化是影响肉牛脂肪沉积和牛肉品质的重要因素之一，解析其机理将助力我国肉牛产业的发展。环状RNA（circRNA）是一类具有重要功能的非编码RNA，参与调控多种生物学过程，但其在牛前体脂肪细胞增殖分化中的研究迄今未见报道。因此，本项目将以秦川牛前体脂肪细胞和成熟脂肪细胞为研究对象，利用高通量测序与生物信息分析，筛选脂肪细胞增殖分化过程中差异表达环状RNA circADs；结合过表达/干扰等策略鉴定4~6个关键circADs；随后利用核质分离技术对circADs进行亚细胞定位，利用流式细胞等技术检测circADs对牛前体脂肪细胞增殖与分化的调控作用，并验证circADs-miRNA-靶基因的互作关系，再通过体外注射小鼠模型验证circADs的功能，最终解析关键circADs对牛前体脂肪细胞增殖分化的调控机制。这些结果将为解析肉牛脂肪增殖分化和改良培育优良肉牛品种提供理论依据。

脂肪细胞的增殖分化是影响肉牛生长发育与肉品质的重要因素之一，对其机理研究有利于解决我国当前地方黄牛品种生长发育缓慢、产肉率低等问题。环形非编码RNA（circular RNA，circRNA）在多个物种的生理功能和病理机制发挥重要调控作用，同样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circRNAs及其功能、调控机制如何，尚不清楚。因此，本项目以秦川牛等为对象，筛选到牛脂肪细胞分化过程中一批差异表达circRNAs，并鉴定出了10个circRNAs（即circAD1～circAD10)。通过实验研究，发现其中9个circRNA（除了circAD5之外）对脂肪细胞增殖分化具有调控作用；并进一步解析了其中circAD1、circAD4、circAD6、circAD7、circAD8、circAD9、circAD10等7个circADs的具体调控机理。此外，还发现circAD1和circAD4分别存在一个SNP位点，功能研究实验阐明这些SNPs通过影响circRNAs的功能来发挥对牛脂肪细胞增殖分化的作用机制。此外，为推进脂肪增殖分化相关ncRNA及靶基因的育种应用，本项目还发现circAD1和circAD4等基因遗传变异与牛体重等性状显著相关，可作为有效的选育遗传标记，为加快肉牛生长发育与牛肉品质改良奠定基础。基于以上研究，本项目已发表SCI 论文11篇，其中 IF>10的 SCI论文1篇，IF>5.0的SCI论文7篇，中科院大类一区SCI论文3篇，中科院大类二区SCI论文5篇；授权国家发明专利2件，申请并受理2件，获批软件著作权2件。