The modification of methyltransferase-like (METTL) enzymes plays important roles in various cellular responses by regulating microRNA expression. However, how m6A modification is involved in stress granule (SG) formation in the early stage of acute ischemic stroke by affecting the biogenesis processing of microRNAs remains unclear. Here, we established a middle cerebral artery occlusion (MCAO) model in rats and an oxygen-glucose deprivation/reperfusion (OGD/R) model in primary cortical neurons and PC12 cells to explore the potential mechanism between m6A modification and SG formation. The in vivo results showed that the level of infarction and apoptosis increased while SG formation decreased significantly within the ischemic cortex with improved reperfusion time after 2 h of ischemia. Consistent with the in vivo data, an inverse association between the apoptosis level and SG formation was observed in PC12 cells during the reperfusion period after 6 h of OGD stimulation. Both in vivo and in vitro results showed that the expression of METTL3 protein, m6A and miR-335 was significantly decreased with the reperfusion period. Overexpression of the METTL3 and METTL3 gene-knockdown in PC12 cells were achieved via plasmid transfection and CRISPR-Cas9 technology, respectively. Overexpression or knockdown of METTL3 in oxygen-glucose deprivation of PC12 cells resulted in functional maturation of miR-335, SG formation and apoptosis levels. In addition, we found that miR-335 enhanced SG formation through degradation of the mRNA of the eukaryotic translation termination factor (Erf1). In conclusion, we found that METTL3-mediated m6A methylation increases the maturation of miR-335, which promotes SG formation and reduces the apoptosis level of injury neurons and cells, and provides a potential therapeutic strategy for AIS.

甲基转移酶样（METTL）酶的修饰通过调节microRNA表达在各种细胞反应中起重要作用。但是，尚不清楚m6A修饰如何通过影响微小RNA的生物发生过程而参与急性缺血性卒中早期的应激颗粒（SG）形成。在这里，我们在大鼠中建立了大脑中动脉闭塞（MCAO）模型，并在原代皮层神经元和PC12细胞中建立了氧葡萄糖剥夺/再灌注（OGD / R）模型，以探索m6A修饰与SG形成之间的潜在机制。的体内结果表明，缺血2 h后，缺血皮层中梗塞和凋亡水平增加，SG形成明显减少，并且再灌注时间延长。与之一致体内数据显示，在OGD刺激6小时后的再灌注期间，PC12细胞中凋亡水平与SG形成之间呈负相关。都体内 和 体外结果表明，METTL3蛋白，m6A和miR-335的表达随着再灌注时间的延长而显着降低。分别通过质粒转染和CRISPR-Cas9技术在PC12细胞中实现了METTL3和METTL3基因敲低的过表达。METTL3在PC12细胞氧葡萄糖剥夺中的过度表达或敲低导致miR-335的功能成熟，SG形成和凋亡水平。此外，我们发现miR-335通过降解真核翻译终止因子（Erf1）的mRNA增强了SG的形成。总之，我们发现METTL3介导的m6A甲基化增加了miR-335的成熟，从而促进SG的形成并降低了损伤神经元和细胞的凋亡水平，并为AIS提供了潜在的治疗策略。