miR-27a参与Myostatin和亮氨酸调节猪成肌细胞增殖分化的功能研究

Our previous work demonstrated that mouse myostatin 3'UTR could be directly targeted by miR-27a and found that the expression pattern of mouse myostatin was opposed to that of miR-27a during mouse C2C12 myoblast proliferation. After analysis, the 3'UTR of porcine myostatin was also observed to contain a single miR-27a target site with complete complementarity in the seed region, indicating that the 3'UTR of porcine myostatin can also be targeted by miR-27a. Based on the hypothesis that reciprocal regulation loop might exit between miR-27a and porcine myostatin and the former might participate in myostatin- and leucine-regulated proliferation and differentiation of porcine myoblast, in this project gene overexpression and gene silencing technologies were employed to regulate specifically miR-27a and porcine myostatin expressions. The aim of this project is to make clear whether reciprocal regulation loop exits between miR-27a and porcine myostatin and to elucidate whether miR-27a participates in myostatin-regulated proliferation and differentiation inhibition of porcine myoblast. On this basis, another aim of this project is to make clear the effect of leucine on porcine myoblast proliferation and differentiation and its miR-27a and myostatin explanatory mechanism. The present project is of importance not only to further understand the mechanisms involved in the control of skeletal muscle size, but also to the study of regulation of pork production and quality by nutrients and its mechanisms.

前期研究确证了小鼠Myostatin为miR-27a的靶基因并发现C2C12细胞增殖过程中小鼠Myostatin与miR-27a表达规律相反。经分析得出猪Myostatin也可能为miR-27a的靶基因。基于"miR-27a与猪Myostatin间存在相互调控作用并以此参与Myostatin抑制猪成肌细胞增殖分化功能"的科学假设，本项目拟采用基因过表达和基因沉默技术特异性调控miR-27a和猪Myostatin的表达，以猪成肌细胞为对象，弄清miR-27a与猪Myostatin之间是否存在相互调控作用，阐明Myostatin抑制猪成肌细胞增殖分化是否需要miR-27a的参与，在此基础上，阐明亮氨基酸对猪成肌细胞增殖分化的影响及miR-27a和Myostatin诠释的作用机理。开展本项目研究，对于进一步认识骨骼肌大小调节机制、深入开展营养对猪肉产量和品质的调控作用及其机制研究具有重要意义。

本项目成功分离出经免疫组化和免疫荧光鉴定过的高纯度的猪成肌细胞。在成功克隆获得猪Myostatin 3'UTR全长序列的基础上，采用荧光素酶报告基因分析方法证实了猪Myostatin为miR-27a的靶基因。在猪心脏和脂肪组织以及猪成肌细胞增殖过程中，miR-27a的表达与猪Myostatin的表达呈负相关。功能研究表明，过表达miR-27a促进了猪成肌细胞的增殖并下调了猪Myostatin的表达，而过表达猪Myostatin抑制了猪成肌细胞的增殖并下调了miR-27a的表达。C2C12细胞上的研究也得到了类似的结果。功能研究同时表明，过表达miR-27a下调了分化过程中细胞内Myogenin的表达，而抑制miR-27a则相反，从而证实了miR-27a具有抑制猪成肌细胞分化的功能。此外，探讨了亮氨酸对猪成肌细胞和C2C12细胞增殖分化的影响及机理，结果表明，亮氨酸可促进细胞增殖、上调miR-27a表达以及下调Myostatin表达，miR-27a抑制剂削弱了亮氨酸的促细胞增殖作用。结果同时表明，亮氨酸可促进猪成肌细胞的分化，并反馈性引起Myostatin的上调。综上结果表明，猪Myostatin与miR-27a间存在相互调控作用，miR-27a具有促进猪成肌细胞增殖并抑制细胞分化的功能并通过其靶基因Myostatin实现，miR-27a可被亮氨酸诱导表达且在亮氨酸促成肌细胞增殖功能中发挥了重要的作用。项目研究结果对进一步认识骨骼肌大小调节机制、深入开展营养对猪肉产量和品质的调控作用及其机制研究具有重要意义。