miR‑335 promotes stress granule formation to inhibit apoptosis by targeting ROCK2 in acute ischemic stroke

Abstract

Under harmful environmental conditions, stress granules (SGs), macromolecular aggregates that are associated with cell survival and death, are produced in the eukaryotic cytoplasm. However, whether and how microRNAs (miRNAs/miRs) modulate SG formation induced by acute ischemic stroke has not been investigated. In the present study, a rat model of middle cerebral artery occlusion (MCAO) was utilized and miRNA array profiling and reverse transcription-quantitative polymerase chain reaction were performed. The results revealed that miR-335 was downregulated during acute ischemic stroke, which was concomitant with reduced SG formation, enhanced apoptosis levels and increased Rho associated protein kinase 2 (ROCK2) expression. In the MCAO rat and serum-free cell models, miR-335 treatment upregulated SG formation, alleviated the ischemia-induced infarction, and decreased ROCK2 protein expression and apoptosis levels. By contrast, when compared with miR-335 treatment, the inhibition of miR-335 resulted in reduced SG formation and higher ROCK2 expression and apoptosis levels. Target prediction analysis and luciferase 3′-untranslated region reporter assay identified ROCK2 as the direct target of miR-335. Furthermore, ROCK2 silencing enhanced SG formation and attenuated the level of apoptosis in the serum-free cell model. In addition, ROCK2 silencing markedly inhibited the effect of miR-335 on SG formation and apoptosis levels. Unexpectedly, the phosphorylation of T-cell intracellular antigen-1 was significantly inhibited by miR-335 in the MCAO rat model, which provides a reasonable explanation for the promotional effect of miR-335 on SG formation by specifically targeting ROCK2. In conclusion, these results demonstrate that miR-335 promotes SG formation and inhibits apoptosis by reducing ROCK2 expression in acute ischemic stroke, which provides a possible therapeutic target for brain injury.

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

“…*P value < 0.05; ***P value < 0.001; ****P value < 0.0001. in the oxygen-glucose deprivation of PC12 cells using doublelabeled immunofluorescence (TIA1 and G3BP1). In our previous publication we showed that SG formation may reduce ischemic cortical injury in the early stage of AIS (Si et al, 2019). Therefore, our in vitro and in vivo experimental results suggested that METTL3-mediated SG formation might reduce AIS injury in the early stage of the disease.…”

“…A total of 108 male Sprague-Dawley (SD: 230-260 g; 8-9-weeks old) rats were randomly divided into six groups (n = 18 rats/group): the sham group (sham operation); R-0 h group (MCAO for 2 h and reperfusion for 0 h); R-6 h group (MCAO for 2 h and reperfusion for 6 h); R-12 h group (MCAO for 2 h and reperfusion for 12 h); R-18 h group (MCAO for 2 h and reperfusion for 18 h); and R-24 h group (MCAO for 2 h and reperfusion for 24 h). The MCAO model was established as previously described (Si et al, 2019). Briefly, after a median incision on the neck, the left common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were isolated.…”

“…In addition, it was found that METTL3 knockout decreased the binding activity between DGCR8 and pri-miRNA, leading to decreased expression of mature miRNAs (Alarcon et al, 2015). Our previous studies showed that miR-335 promotes the production of stress granules by targeting the Rho-associated coiled-coil forming protein kinase 2 (ROCK2), and its expression is reduced in AIS specificity (Si et al, 2019). Therefore, we hypothesized that m6A modification is involved in SG formation in the early stage of AIS by affecting the biogenesis processing of miR-335.…”

RNA Binding Protein Motif 3 Inhibits Oxygen-Glucose Deprivation/Reoxygenation-Induced Apoptosis Through Promoting Stress Granules Formation in PC12 Cells and Rat Primary Cortical Neurons

“…Emerging evidence suggests that SGs formation and ischemia-reperfusion onset may occur concurrently in principle because of the similarity in the two events ( Lee and Seydoux, 2019 ; Wolozin and Ivanov, 2019 ). Our previous research demonstrates that the marker of stress granules [T-cell intracellular antigen 1 (TIA1)] is upregulated at the early time-points of blood reperfusion after 2 h of cerebral ischemia, accompanying a significant decrease of apoptosis level in the ischemic cortex ( Si et al, 2019 ). This finding hint that SGs formation may occur in the early state of AIS, but the direct evidence is still lacking.…”

“…AIS triggers a damaging cascade as a multifactorial disease, including endoplasmic reticulum (ER) stress, oxidative stresses, excitotoxicity, inflammation, and apoptosis ( Bettencourt and Ferro, 2020 ; Sui and Gao, 2020 ). Notably, ischemic cascade conditions exhibit very similar characteristics to stress granules (SGs) formation ( Si et al, 2019 ). Stress granules are stalled translational aggregates generated in the cytoplasm and induced by varied environmental stress, including heat shock, hypoxia, ER stress, and oxidative stress ( An et al, 2019 ; Guzikowski et al, 2019 ; Ivanov et al, 2019 ).…”