Fear stress during pregnancy affects placental m6A-modifying enzyme expression and epigenetic modification levels. Rattus norvegicus

As the hub connecting mother and offspring, the placenta's normal development is vital for fetal growth. Fear stress can cause some structural alterations of the placenta and affect placental development and function.N6-methyladenosine (m6A) is the most abundant modification in mRNA, which can be involved in the regulation of placenta and embryo development. There are no reports regarding the potential role of the m6A modification in placental damage caused by fear stress during pregnancy. In this study, fear stress during pregnancy leads to the rise of methylated enzymes (METTL3, METTL14, WTAP), the decrease of the demethylase FTO, and the increase of the overall methylation level placenta of pregnant rats. MeRIP-seq data analysis showed 22,010 m6A peaks associated with 12,219 genes in the placenta of the model and 21,060 m6A peaks associated with 11,730 genes in the placenta of the control. The peaks were mainly concentrated in the coding region and the 3'UTR. In addition, 50 genes with abnormal modification and expression(double aberrant genes) were screened out by combining MeRIP-seq and RNA-seq data. Mefv, Erbb2, and Cgas were selected from 50 double aberrant genes, and MeRIP-qPCR and RT-qPCR were used to verify modification and expression levels. This suggests that m6A modifications may play an important role in fear stress induced placental dysfunction during pregnancy.

胎盘作为连接母体与子代的枢纽，其正常发育对胎儿生长发育至关重要。恐惧应激可引发胎盘结构改变，并影响胎盘的发育与功能。N6-甲基腺嘌呤（N6-methyladenosine，m6A）是mRNA中含量最为丰富的修饰形式，可参与调控胎盘与胚胎的发育过程。目前尚无关于m6A修饰在妊娠期恐惧应激所致胎盘损伤中潜在作用的研究报道。本研究中，妊娠期恐惧应激可使孕鼠胎盘的m6A甲基化酶（METTL3、METTL14、WTAP）表达上调，去甲基化酶FTO表达下调，并导致胎盘整体m6A甲基化水平升高。甲基化RNA免疫沉淀测序（MeRIP-seq）数据分析显示，模型组胎盘中共检测到22010个m6A峰，关联12219个基因；对照组胎盘中共检测到21060个m6A峰，关联11730个基因。这些m6A峰主要富集于编码区及3'非翻译区（3'UTR）。此外，联合MeRIP-seq与RNA测序（RNA-seq）数据分析，共筛选得到50个修饰与表达均异常的基因（双重异常基因）。从50个双重异常基因中筛选出Mefv、Erbb2及Cgas，并通过甲基化RNA免疫沉淀定量PCR（MeRIP-qPCR）与逆转录实时定量PCR（RT-qPCR）验证了其修饰水平与表达量。上述结果表明，m6A修饰可能在妊娠期恐惧应激诱导的胎盘功能异常中发挥重要调控作用。