Methyltransferase 3 Mediated miRNA m6A Methylation Promotes Stress Granule Formation in the Early Stage of Acute Ischemic Stroke

Abstract

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.

Subcellular Localization of miRNAs and Implications in Cellular Homeostasis

“…Further, miR-335 directly targeted the ROCK2 3 UTR to inhibit apoptosis in PC12 cells [104]. Interestingly, in ischemic cortices of MCAO rats, METTL3, m6A, and miR-335 were up-regulated at 0 h after reperfusion but significantly down-regulated at 24 h after reperfusion [104]. This was similar in PC12 cells treated with oxygen-glucose deprivation/reperfusion (OGD/R) stimulation as an in vitro model of acute ischemic stroke [104].…”

“…Interestingly, in ischemic cortices of MCAO rats, METTL3, m6A, and miR-335 were up-regulated at 0 h after reperfusion but significantly down-regulated at 24 h after reperfusion [104]. This was similar in PC12 cells treated with oxygen-glucose deprivation/reperfusion (OGD/R) stimulation as an in vitro model of acute ischemic stroke [104]. Furthermore, METTL3-mediated m6A modifications of pri-miR-335 promoted the mature miR-335 expression, enhancing SG formation and attenuating apoptosis in OGD/R stimulated PC12 cells.…”

“…Conversely, in acute ischemic stroke rats model of middle cerebral artery occlusion (MCAO), miR-335 promoted SG formation by downregulating ROCK2 [103]. Further, miR-335 directly targeted the ROCK2 3 UTR to inhibit apoptosis in PC12 cells [104]. Interestingly, in ischemic cortices of MCAO rats, METTL3, m6A, and miR-335 were up-regulated at 0 h after reperfusion but significantly down-regulated at 24 h after reperfusion [104].…”

Alteration of N6 -Methyladenosine mRNA Methylation in a Rat Model of Cerebral Ischemia–Reperfusion Injury

“…It has been reported that m6A levels in the cortex and hippocampus of Alzheimer's disease (AD) mice were notably higher than those in the control group and that m6A methylation genes were involved in the pathways associated with the occurrence of AD (Han et al, 2020). Si et al (2020) found that METTL3-mediated miRNA m6A methylation promotes stress granule formation and reduces the apoptosis of injury neurons in the early stage of acute IS. Furthermore, Wang et al (2020a,b) found that the expression levels of METTL3 and WTAP were reduced in the larger pathological tissue of cerebral AVMs.…”

Regulatory Mechanisms of the RNA Modification m6A and Significance in Brain Function in Health and Disease

“…Using the middle cerebral artery occlusion (MCAO) model of ischemic stroke in rats, Si and colleagues found that METTL3-mediated methylation promoted maturation of miR-355 which then promoted stress granule formation which was neuroprotective ( Si et al, 2020 ). A role for m 6 A readers as regulators of the brain response to stroke has also been proposed.…”