Preliminary Investigation Of The Mechanism Of LncRNA MIAT Promoting Cerebral Ischemia Reperfusion Injury

ObjectiveTo investigate the effect of LncRNA MIAT on cerebral ischemia-reperfusion injury through cerebral ischemia-reperfusion model in vivo and in vitro and to elucidate the mechanism preliminarily.Methods(1)An in vitro model of cerebral ischemia-reperfusion was constructed with oxygen-glucose deprivation/ reperfusion(OGD/R)treatment on SH-SY5 Y cells.The effects of OGD/R treatment on SH-SY5 Y cells activity were detected by CCK-8 assay and the effect of LncRNA MIAT expression were detected by RTqPCR.(2)Observe the distribution of LncRNA MIAT in SH-SY5 Y cells using RNA fluorescence in situ hybridization(FISH).(3)Transfect SH-SY5 Y cells with si-MIAT to down-regulate LncRNA MIAT and verify the effect of downregulation by RT-qPCR.(4)Observe the changes in autophagy of SH-SY5 Y cells after downregulation of LncRNA MIAT from mRNA,protein and morphology perspectives using RT-qPCR,Western Blotting and transmission electron microscopy,respectively.(5)Simulation of changes in cerebral ischemiareperfusion in vivo using the MCAO/R model in C57BL/6J mice.(6)Silencing LncRNA MIAT in mice with adeno-associated virus(AAV)and verifying the silencing efficiency by RT-qPCR.(7)Assess the degree of neurological deficits in the MCAO/R mouse model before and after LncRNA MIAT silencing using the Longa scale.(8)Compare the effects of LncRNA MIAT silencing on the size of MCAO/R-induced cerebral infarct area before and after silencing by TTC staining.(9)Observe the effect of LncRNA MIAT silencing on MCAO/R-induced morphological changes of brain tissue before and after silencing by HE staining.(10)Verify the expression changes of p62,LC3 and Beclin-1 in MCAO/R mouse models before and after LncRNA MIAT silencing in vivo by rt-PCR and Western Blotting.ResultsLncRNA MIAT was expressed in both cytoplasm and nucleus of SH-SY5 Y.OGD/R treatment decreased the activity of SH-SY5 Y cells and significantly increased LncRNA MIAT expression,while p62 expression was decreased and LC3 expression was increased,suggesting that its autophagy level was upregulated.The efficiency of silencing LncRNA MIAT by transfection with siMIAT in vitro was about 50%.Silencing of LncRNA MIAT slowed down the trend of increased autophagy level of SH-SY5 Y caused by OGD/R treatment.MCAO/R treatment increased LC3 and Beclin-1 levels and decreased p62 in mice with severe brain tissue damage and increased neurological deficit scores,whereas silencing LncRNA MIAT with AAV resulted in reduced brain infarct size and decreased neurological deficit scores after MCAO/R.Compared with the control group,silencing LncRNA MIAT slowed the trend of increased LC3 and Beclin-1expression and decreased p62 expression caused by MCAO/R.Conclusion(1)LncRNA MIAT was expressed in both nucleus and cytoplasm of SHSY5 Y cells.In SH-SY5 Y cells,OGD/R treatment reduced cell activity accompanied by increased autophagy levels and increased LncRNA MIAT expression.Down-regulation of LncRNA MIAT in SH-SY5 Y cells attenuated the trend of increased autophagy levels caused by OGD/R.(2)MCAO/R caused brain tissue damage and neurological deficits and increased autophagy levels in vivo,while down-regulation of LncRNA MIAT attenuated the trend of brain tissue damage and increased autophagy levels caused by MCAO/R.