METTL3介导的ATG5 m6A修饰在妊娠期糖尿病胎盘中的作用及机制研究

The changes of internal environment in gestational diabetes mellitus (GDM) that influence trophoblast functions may lead to several pregnancy complications and adverse pregnancy outcomes, however, the mechanism underlying high glucose regulates trophoblast functions is still unclear. We have previously reported that levels of ATG5 and autophagy were aberrantly enhanced, and recently we find decreased levels of METTL3 and m6A modification in the placenta of GDM and high glucose treated trophoblast cell line (HTR8/SVneo). Based on these findings, we will illustrate how METTL3 influences autophagy and functions of trophoblasts firstly. Then the mechanisms of ATG5 and autophagy regulated by METTL3-mediated m6A modification will be demonstrated by the combination of epitranscriptomic and translatomic analysis. RNA Binding Protein Immunoprecipitation will be performed to further investigate whether the stability of ATG5 mRNA is regulated by YTHDF2 through recognition m6A modification. Finally, GDM mice are used to illustrate the function of METTL3 in placental and fetal development. This study aims to demonstrate the mechanisms of m6A in regulating trophoblast functions through autophagy, which will provide novel insights for researches focused on high glucose mediated RNA posttranscriptional modifications and epigenetic regulation of autophagy, and provide novel targets for the control of GDM.

妊娠期糖尿病（GDM）患者内环境改变对胎盘滋养层细胞的影响可能是导致严重的妊娠并发症和不良妊娠结局的重要原因之一，但高糖调控滋养层细胞功能的分子机制尚未明确。本课题组前期发现GDM患者胎盘及高糖环境下滋养层细胞系（HTR8/SVneo）中ATG5和自噬水平升高，近期结果显示RNA甲基化酶METTL3及m6A水平降低。本项目拟在此基础上，以HTR8/SVneo为体外模型，研究METTL3对滋养层细胞自噬和功能的影响，联合表观转录组和翻译组探讨METTL3介导的m6A修饰对ATG5和自噬的调控机制，利用RNA结合蛋白免疫沉淀研究YTHDF2识别m6A修饰对ATG5稳定性的调控，最后研究METTL3对GDM小鼠胎盘及胎鼠的影响。本研究旨在阐明高糖环境下m6A调控自噬影响滋养层细胞功能的作用机制，可为高糖调节RNA转录后修饰、表观修饰调控自噬的研究提供新线索，为GDM的防治探寻新的分子靶点。

妊娠期糖尿病（GDM）患者内环境改变对胎盘滋养层细胞的影响可能是导致严重的妊娠并发症和不良妊娠结局的重要原因之一，但高糖调控滋养层细胞功能的分子机制尚未明确。本课题组前期发现GDM患者胎盘及高糖环境下滋养层细胞系中ATG5和自噬水平升高，近期结果显示RNA甲基化酶METTL3及m6A水平降低。在此基础上，我们利用多种分子生物学技术开展研究，探讨高糖环境下m6A调控自噬影响滋养层细胞功能的作用机制。结果显示：敲低METTL3导致滋养层细胞增殖降低、凋亡增加、侵袭能力减弱。MeRIP-seq显示沉默METTL3导致ATG5 m6A修饰水平降低，其降解速率下降而稳定性升高。核糖体新生肽链复合体测序（RNC-seq）显示沉默METTL3提高了大部分自噬调控基因的mRNA翻译水平。通过RNA免疫共沉淀试验，证明m6A reader 蛋白YTHDF1/2能够直接与ATG5的mRNA结合，并且沉默METTL3后YTHDF1与ATG5的mRNA结合能力升高，而YTHDF2与ATG5的mRNA结合能力下降，两者分别通过促进翻译和抑制降解，从而导致ATG5蛋白水平的升高，以及自噬水平的增加。本项目将为高糖环境下m6A调控自噬影响滋养层细胞功能的作用机制提供新线索，为GDM的防治探寻新的分子靶点。