延边黄牛和韩延牛血液外胞体中与垂体GH调控相关的差异miRNA筛选及功能研究

There are slow growth, long growth cycle and low slaughter rate during the breeding process of Yanbian yellow cattle, which seriously restricts the rapid development of Yanbian yellow cattle breeding industry. In recent years, the miRNA found in the Exosome of animal can regulate the growth of animal and is one of the important hotspot research at home and abroad. Although certain progress has been achieved on the regulation of miRNA in the Exosome on animal growth at home and abroad, the research of regulation of the miRNA in the Exosome on growth of Yanbian yellow cattle has not been reported. The miRNA will be separated from the blood Exosome of the Yanbian yellow cattle and Hanyan cattle in this study, and the expression characteristics of miRNA in the blood Exosome of Yanbian yellow cattle and Hanyan cattle will be analysized through RNASEQ sequencing and bioinformatics software technology and also find out different expression miRNA closely associating with pituitary growth hormone (GH). The reporter gene, cell model analysis and in vivo validation methods will be used to determine the target gene of the different expression miRNA and their regulation mechanism about pituitary GH of Yanbian yellow cattle. This study will not only accumulate and provide a theoretical basis for the epigenetic modification of growth and development of Yanbian yellow cattle, but also provide a new target for the regulation of growth and development of Yanbian yellow cattle.

延边黄牛生长速度慢、生长周期长和出栏率低等缺点严重制约着延边黄牛养殖业的快速发展。近年来发现，动物体内外胞体（Exosome）中的miRNA具有调控动物生长的功能，是国内外研究的重要热点之一。虽然国内外对Exosome中miRNA调控动物生长的研究取得了一些进展，但关于Exosome中miRNA对延边黄牛生长调控方面的研究国内外均未见报道。本研究拟从延边黄牛和韩延牛血液Exosome中分离miRNA，通过RNASEQ测序和生物信息学软件技术分析延边黄牛及韩延牛血液Exosome中miRNA的表达特点，并寻找出与垂体生长激素（GH）密切相关的差异表达miRNA。采用报道基因、细胞模型分析和活体验证等方法，确定差异表达miRNA调控的靶基因及对延边黄牛垂体GH的调控机制。本课题的研究，不仅为延边黄牛生长发育的表观遗传修饰积累和提供理论依据，也为延边黄牛生长发育调控提供新的作用靶标。

如何提高纯种延边黄牛的生长速度、缩短其养殖周期是加快延边黄牛养殖产业快速发展的关键。研究表明外胞体（exosome）中miRNA在动物的生长调控方面发挥着重要作用，但Exosome中miRNA是如何通过调控垂体生长激素（GH）合成而调控延边黄牛生长速度的有待进一步研究。因此本项目选择以延边黄牛与韩延牛作为研究对象，研究了延边黄牛及韩延牛血液Exosome中miRNA的表达特点，寻找与垂体生长激素（GH）相关的差异表达miRNA，并在细胞水平和活体水平上研究了其对延边黄牛腺垂体GH的调控机制。研究发现，Exosome中主要有5个关键差异表达miRNA（miR-10b、miR-6523a、miR-93、miR-23b-3p、miR-1468）与垂体GH的合成相关。其中，miR-10b和miR-6523a主要通过分别抑制SSTR2和SSTR5靶基因蛋白表达而提高垂体GH的含量，而miR-93、miR-23b-3p和miR-1468通过分别抑制GHRHR、POU1F1和CREB1靶基因蛋白表达而降低垂体GH含量，进而调控延边黄牛的生长速度。本项目的研究成果将为延边黄牛生长发育的表观遗传修饰积累和提供理论依据，也为延边黄牛生长发育调控提供新的作用靶标。