The protective effects of lncRNA ZFAS1/miR-421-3p/MEF2C axis on cerebral ischemia-reperfusion injury

LncRNA ZNFX1 antisense RNA 1 (ZFAS1) could improve neuronal damage and inhibit inflammation and apoptosis. We conducted an in-depth exploration on the protective mechanism of ZFAS1 in cerebral ischemia-reperfusion injury. Overexpressed or silenced plasmids of ZFAS1 were transfected into the cells to analyze the effects of oxygen-glucose deprivation/reperfusion (OGD/R) treatment on the viability, apoptosis and related gene expressions of Neuro-2a cell by performing MTT assay, flow cytometry, qRT-PCR, and Western blot. Bioinformatic analysis, qRT-PCR, dual-luciferase reporter assay and RNA immunoprecipitation were used to screen and verify the miRNA(s) which could competitively bind with ZFAS1 and downstream mRNA(s) targeted by the miRNA(s). The effects of ZFAS1 and the above target miRNA(s) or gene(s) on the apoptosis of OGD/R-injured cells, apoptosis-related proteins, inflammatory factors and p65/I kappa B alpha pathway were further verified via the rescue test. The results from the middle cerebral artery occlusion (MCAO) mouse model in vivo were consistent with those from the cellular experiments. The expression of lncRNA ZFAS1 in OGD/R-injured cells was inhibited, and the up-regulation of ZFAS1 protected Neuro-2a cells. MiR-421-3p was predicted to be the target miRNA of ZFAS1 and could offset the protective effect of ZFAS1 overexpression on OGD/R-injured cells following its up-regulation. MEF2C, which was the downstream target gene of miR-421-3p, reversed the OGD/R-induced enhanced cell damage caused by miR-421-3p mimic when MEF2C was overexpressed. In in vivo studies, ZFAS1 overexpression reduced brain tissue infarction, apoptosis and gene regulation caused by MCAO, while miR-421-3p mimic had the opposite effect. Collectively, the regulation of lncRNA ZFAS1/miR-421-3p/MEF2C axis showed protective effects on cerebral ischemia-reperfusion injury.

Automated summary

The present study investigated the protective mechanism of ZFAS1 in cerebral ischemia-reperfusion injury. The up-regulation of ZFAS1 was found to improve cell damage, inhibit apoptosis, and reduce inflammation. MiR-421-3p was identified as a target miRNA of ZFAS1 and its up-regulation attenuated the protective effect of ZFAS1 overexpression. MEF2C, the downstream target gene of miR-421-3p, reversed the enhanced cell damage caused by miR-421-3p mimic when overexpressed. In vivo experiments confirmed the protective effects of ZFAS1 overexpression in reducing brain tissue infarction, apoptosis, and gene regulation.