RNA甲基化（m6A）甲基转移酶VIRMA通过调控Rassf5表达影响植入后胚胎发育的机制研究

Embryo aresst seriously affects female reproductive and mental health, but the mechanism needs to be further studied. VIRMA is a key methyltransferase of RNA m6A, and its deletion causes death after implantation of mouse embryos. The specific mechanism has not been resolved. Applicant has previously obtained Virma+/- mice and confirmed the lethality of their homozygous deletions, and screened for a downstream key target gene Rassf5 associated with cell development. On this basis, the project aims to analyze the expression of Rassf5 by knocking out and over-expressing Virma in MEF cells, and localizing Virma and Rassf5 in the embryo by RNA in situ hybridization. Embryos at different time points after implantation were obtained by Virma+/- mouse hybridization. Genotype identification and statistical analysis confirmed the timing of embryonic death caused by Virma deletion, and analyzed the apoptosis and proliferation of Virma-/- and wild-type embryonic cells, revealing the specific mechanism of action of VIRMA, and the analysis of MeRIP-seq and RNA-seq, were performed to screen for other target genes and further verify their specific functions.. Therefore, the mechanism of RNA methylation in embryonic cessation can be preliminarily explored, which will help to deepen the understanding of embryo development after implantation and provide new ideas for embryonic prevention and treatment.

胚胎停育严重影响女性生殖和心理健康，但是发生机理有待深入研究。VIRMA作为RNA m6A的关键甲基转移酶，其缺失会造成小鼠胚胎植入后死亡，具体机制尚未得到解析。申请人前期已获得Virma+/-小鼠并证实了其纯合缺失致死性，并筛选出与细胞发育相关的下游关键靶基因Rassf5。在此基础上本项目拟通过敲降和过表达MEF细胞中的Virma，分析Rassf5表达变化，并通过RNA原位杂交对胚胎中Virma及Rassf5进行定位。通过Virma+/-小鼠杂交获得植入后不同时间点胚胎，基因型鉴定及统计分析确定Virma缺失造成胚胎死亡的时间，分析Virma-/-与野生型胚胎细胞凋亡及增殖情况，揭示VIRMA的作用机制，并进行MeRIP-seq和RNA-seq分析，筛选其他靶基因并验证。从而初步探讨RNA甲基化在胚胎停育中的作用机制，有助于加深对人植入后胚胎发育的认识，为胚胎停育防治提供新思路。

胚胎停育严重影响女性生殖和心理健康。解析胚胎停育的具体原因不仅有助于提升辅助生殖的成功妊娠率，而且可以减少流产率，优化辅助生殖体系。然而，目前对于胚胎停育具体调控机制的认识非常有限，有待进一步研究。VIRMA作为RNA m6A的关键甲基转移酶，其缺失会造成小鼠胚胎植入后死亡，具体机制尚未得到解析。本项目主要内容为利用Virma+/-小鼠明确Virma纯合缺失的致死时间点，并深入解析致死之前WT和KO胚胎转录组差异，揭示关键致死机制。研究发现，Virma缺失后关键致死时间点为囊胚至E6.5阶段，因此Virma纯合缺失引起植入失败。我们利用体外培养及单细胞MALBAC检测获得WT和KO囊胚，活检后进行SMART-seq2单细胞转录组测序，发现KO组内细胞团和外滋养层细胞转录活性异常增强，大量基因表达显著升高。通过GO分析，发现上调的基因显著富集了“线粒体基因表达”和“线粒体核糖体组装”两个基因Term，由此我们推测Virma的缺失造成了线粒体功能的异常最终引起细胞无法增殖分化完成胚胎发育。本项目数据首次从胚胎期致死基因出发，明确致死时间点，研究了RNA m6A的关键甲基转移酶VIRMA在胚胎发育中的关键功能。研究结果将有助于加深对人植入后胚胎发育的认识，为胚胎停育防治提供新思路。