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.

Keywords: Erf1; METTL3; acute ischemic stroke; miR-335; stress granules.

FIGURE 1

The inverse relation between SG formation and ischemic injury in ischemic cortex of MCAO rats. (A) Infarct volume was the greatest after 24 h of reperfusion in the MCAO model. TTC staining was used to measure infarct size (n = 72 rats, 12 rats/group). (B) The most severe brain injury appeared at 24 h post-reperfusion. (C) Apoptosis levels were elevated in the ischemic cortices of MCAO rats after 24 h of reperfusion. Green color represents apoptotic cells, and blue color represents DAPI staining for the nucleus. (D) SG formation was increased in the MCAO model after 0 and 6 h of reperfusion. SG labeled with TIA1 (green), G3BP1 (red), and DAPI (blue). Scale bars: 10 and 25 μm. SG, stress granules; MCAO, middle cerebral artery occlusion. **P value < 0.01; ****P value < 0.0001.

FIGURE 2

Expression levels of METTL3, m6A, and miR-335 in ischemic cortices of MCAO rats at different times of reperfusion (n = 36 rats, 6 rats/group). (A) Expression levels of METTL3 in the cortices of MCAO rats at different times of reperfusion. Rat cortices were obtained at different times of reperfusion (0, 6, 12, 18, and 24 h) after 2 h of MCAO surgery. (B) m6A levels of total RNA in cortices of MCAO rats at different times of reperfusion. (C) Pri-, Pre, and Mature-miR-335 expression levels in cortices of MCAO rats at different times of reperfusion. m6A, N6-methyladenosine. *P value < 0.05; **P value < 0.01; ***P value < 0.001; ****P value < 0.0001.

FIGURE 3

The inverse relation between SG formation and apoptosis levels in PC12 cells and primary cortical neurons under OGD/R stimulation. (A) SG formation in primary cortical neurons treated with different times of reperfusion (0, 6, 12, 18, and 24 h) after 6 h of OGD stimulation. SG labeled with TIA1 (green), G3BP1 (red), and DAPI (blue). (B) SG formation in PC12 cells treated with different times of reperfusion (0, 6, 12, 18, and 24 h) after 6 h of OGD stimulation. (C) Apoptosis levels in PC12 cells treated with different times of reperfusion (0, 6, 12, 18, and 24 h) after 6 h of OGD stimulation. Scale bars: 10 μm. ****P value < 0.0001.

FIGURE 4

Expression levels of METTL3, m6A and miR-335 in PC12 cells at different times of reperfusion. (A) Expression levels of METTL3 in PC12 cells at different times of reperfusion. PC12 cells were treated with different times of reperfusion (0, 6, 12, 18, and 24 h) after 6 h of OGD stimulation. (B) m6A levels of total RNA in PC12 cells at different times of reperfusion. (C) Pri-, Pre-, and Mature-miR-335 expression levels in PC12 cells at different times of reperfusion. *P value < 0.05; **P value < 0.01; ***P value < 0.001; ****P value < 0.0001.

FIGURE 5

METTL3 promotes the m6A level of pri-miR-335, mature-miR-335, and SG formation in PC12 cells under OGD stimulation. (A) The efficiency of METTL3 knockdown or overexpression in PC12 cells. Western blot assays were used for validation. (B) m6A levels of total RNA in PC12 cells at different times of reperfusion. PC12 cells were treated for different times of reperfusion (0, 6, 12, 18, and 24 h) after 6 h of OGD stimulation. (C) The m6A level of pri-miR-335 in METTL3 knockdown or overexpression PC12 cells under OGD stimulation. (D) The expression level of pri-, pre-, and mature-miR-335 in METTL3 knockdown or overexpression PC12 cells under OGD stimulation. (control-OGD vs. METTL3-OE-OGD; P < 0.001; control-OGD vs. METTL3-KD-OGD; P < 0.0001). (E) SG formation in METTL3 knockdown or overexpression PC12 cells under OGD stimulation (control vs. METTL3-OE; P < 0.001; control vs. METTL3- KD; P < 0.001). *P value < 0.05; **P value < 0.01; ***P value < 0.001; ****P value < 0.0001.

FIGURE 6

MiR-335 specifically decreases Erf1 expression, promotes SG formation, and prevents cell apoptosis levels in PC12 cells under OGD stimulation. (A) MiR-335 directly targeted the 3′-UTR of Erf1 mRNA in PC12 cells. The results of the dual-luciferase reporter system assay are shown in figure. (B) Erf1 protein expression was decreased in PC12 cells transfected with miR-335 mimic. (C) Efficiency of Erf1 knockdown in PC12 cells. (D) SG formation was elevated in Erf1-knockdown PC12 cells under OGD stimulation. Cells were stimulated for 6 h under OGD conditions. (E) The apoptosis level was reduced in Erf1-knock PC12 cells under OGD stimulation. Annexin V-FITC/PI double staining assay was used for apoptosis levels of cells detection. X axis: PI; Y axis: Annexin V. control-OGD vs. Erf1-KD-OGD; P < 0.05. *P value < 0.05; ***P value < 0.001; ****P value < 0.0001.