miR-302/367 cluster对绵羊体细胞核移植效率的影响及其机理研究

Embryonic stem cell-specific cell cycle-regulating microRNAs (ESCC miRNAs) have the ability to improve the efficiency of induced pluripotent stem cells (iPSCs), can even directly reprogram somatic cells into iPSCs. Somatic cell nuclear transfer (SCNT), another type of technology, can also be used to reprogram somatic cells. However, it is unclear whether ESCC miRNAs can be used to improve the efficiency of SCNT. In the present project, sheep-specific ESCC miRNAs (miR-302/367 cluster) will be delivered into sheep skin fibroblasts (SSFs), then, transgenetic SSFs will be used as nuclear donors to construct sheep SCNT embryos. Subsequently, the developmental ability and the quality of SCNT-derived embryos, as well as the pattern in gene expression and epigenetic modifications in these SCNT-derived embryos, will be detected, the effect of ESCC miRNAs on the efficiency of SCNT will be explored. Moreover, the mechanism of ESCC miRNAs affecting the efficiency of SCNT will be explored at the levels of cell, transcription and epigenetic modifications. The results of this project can be used to found a theoretical and technical foundation for improving the efficiency of SCNT by ESCC miRNAs, make it possible for application of SCNT technology in the fields of animal husbandry and regenerative medicine.

胚胎干细胞特异性细胞周期调控microRNAs（ESCC miRNAs）可促进甚至直接诱导体细胞再程序化为诱导性多潜能干细胞（iPSCs）。体细胞核移植（SCNT）是另一种体细胞再程序化技术，然而，ESCC miRNAs能否提高该技术的成功率还未见报导。本项目拟将绵羊特异性的ESCC miRNAs（miR-302/367 cluster）导入绵羊皮肤成纤维细胞中，随后，以转基因细胞为核供体，采用SCNT技术制备绵羊体细胞核移胚，通过检测核移胚的发育率、质量、基因表达模式及表观遗传修饰模式，探究ESCC miRNAs对SCNT效率的影响。在此基础上，在细胞水平、转录水平及表观遗传修饰水平上探究ESCC miRNAs影响SCNT效率的机理。本项目的研究成果可为采用ESCC miRNAs提高SCNT技术的成功率奠定理论及技术基础，使该技术能够真正应用于畜牧业及再生医学领域。

miR-302/367 cluster可促进甚至直接诱导小鼠及人的多种类型体细胞重编程为诱导性多潜能干细胞。然而，其能否提高体细胞核移植（SCNT）技术的总体成功率还未见报导。本项目采用可诱导表达的质粒载体携带绵羊miR-302/367 cluster，采用脂质体转染的方式将重组质粒载体导入绵羊皮肤成纤维细胞（SSFs）中。随后，在细胞水平、转录水平及表观遗传修饰水平上探究miR-302/367 cluster对SSFs增殖、凋亡、细胞周期、基因表达及表观遗传修饰模式的影响。在此基础上，以转基因及非转基因SSFs为核供体，采用SCNT技术制备绵羊体细胞核移胚，通过检测核移胚在发育率及质量方面的差异，确定miR-302/367 cluster在SSFs中预表达对绵羊体细胞核移植效率的影响。研究结果表明，miR-302/367 cluster可作用于CDK2、E2F1、E2F2及PTEN mRNA的3’端非编码序列，导致这些基因的mRNA被降解或其翻译被抑制，最终导致转基因SSFs中PI3K-Akt信号通路被激活，细胞周期信号通路被抑制，降低了转基因SSFs的增殖率及凋亡率。此外，miR-302/367 cluster可下调DNMT1、DNMT3a、DNMT3b、AOF1及AOF2的表达量，上调HDAC1及HDAC2的表达量，导致转基因SSFs中较低的DNA甲基化修饰水平及较高的组蛋白H3乙酰化修饰水平。体细胞核移胚的发育结果表明，miR-302/367 cluster在SSFs中预表达可显著提高绵羊体细胞核移胚的发育率及胚胎质量。本项目从机理上阐明了miR-302/367 cluster对SSFs增殖、凋亡、细胞周期、基因表达及表观遗传修饰模式的影响，为采用miR-302/367 cluster提高绵羊SCNT技术的总体成功率奠定了理论及技术基础。