小鼠胚胎基因组激活中miRNAs表达的表观遗传调节及其作用研究

miRNAs play important roles in mammalian zygotic gene activation after fertilization and in early embryo development, but the role of miRNAs during global DNA demethylation and its expression regulation remains to be elucidated. In this research project, firstly, the different expression of miRNAs was detected by miRNA microarray or sequencing between 2-cell block embryo or non-block mouse embryos fertilized in vitro. After screening the Differentially expressed miRNAs candidates, their function on embryo development and their predicted target genes were validated. Secondly, by comparing the DNA methylation and the histone modification in the regulate region of the candidate miRNAs, combining with DNA demethylation reagents treatment, to examine the effect of epigenetic modifications on the role of miRNAs expression, and its role on embryonic genome activation; In addition, a set of miRNAs related to maternal RNA clearance, ZGA, and cell pluripotency and their target genes were chosen to examined their dynamic expression and epigenetic regulation between the two group of embryos. The purpose of this study is to clarify the regulation of miRNA and its impact on embryonic development and cellular features, to explore the methods for identification of embryo developmental potential by using miRNA as marker, this study also provides a foundation for optimization of the assisted reproductive technologies, such as in vitro fertilization, and somatic cell nuclear transfer.

哺乳动物受精卵中miRNAs的种类、作用靶标及自身表达调节在胚胎基因组激活和细胞重编程中的作用有待研究。本研究采用胚胎发育阻滞模型，比较2-细胞阻滞和非阻滞胚胎miRNAs表达差异，筛选和验证miRNAs功能及对预测靶基因的作用，获得发育相关候选miRNAs；通过比较候选miRNAs转录调节区DNA甲基化和组蛋白修饰在两组胚胎间的差异，结合药物处理，检测表观遗传修饰对miRNAs表达的作用，探讨表观重编程与miRNAs自身调节的相互关系及对胚胎基因组激活的影响；同时，选择以母源RNA清除、合子基因组激活及多能性相关miRNAs及其靶标基因，比较其在两组胚胎间的差异调节，对比检测培养液中胚胎表达的miRNAs成分，建立以miRNAs为标记的胚胎发育潜能鉴定方法。本研究旨在阐明重编程中miRNAs自身调节及对胚胎发育和细胞特性的影响机制，为体外受精、核移植等辅助生殖技术的优化提供依据。

MicroRNAs（miRNAs）是一类长度约为 22 核苷酸的非编码 RNAs，miRNAs 可参与调控多个生物学过程。有研究表明，miR-290 家族成员具有维持胚胎干细胞ESCs多能性的作用。本研究利用显微注射、RT-qPCR、荧光原位杂交、免疫印迹杂交和荧光素酶报告系统等方法研究了 miR-290 家族在植入前胚胎合子基因组激活中表达动态、作用及其上下游调控机制展开研究，旨在阐明重编 程中miRNAs自身调节及对胚胎发育和细胞特性的影响机制，为体外受精、核移植等辅助生殖技术的优化提供依据。研究结果表明，miR-290 家族在胚胎发育在具有重要的作用，该家族的表达在上游受到多能性转录因子 Oct-4 的调控，下游通过直接结合细胞周期抑制基因 p21 mRNA 的3ꞌ 非翻译区调控其表达，参与调控合子基因组激活。另外，在胚胎阻滞模型中，ZGA 标志基因和 miR-290 家族成员的表达均显著下降，表明 miR-290 家族成员的表达可用于鉴定胚胎发育潜能。