Roles Of LncRNA-1810034E14Rik In Modulating Functions Of Microglial Cells After Ischemic Stroke And The Underlying Mechanisms

Background:Neuro-inflammation induced by ischemic stroke directly affects prognosis of patients.Activation status and function of microglia regulate neuro-inflammation and affect the outcome of ischemic stroke.It has been reported that a variety of long non-coding RNAs(lncRNAs)differentially expressed in microglia after ischemic stroke.These IncRNAs may be involved in regulating the function of microglia,but the specific mechanism has not been clarified.LncRNA-1810034E14Rik is widely distributed in microglia,but the function of lncRNA-1810034E14Rik is still unknown.In this study,we discussed the change of lncRNA-1810034E14Rik after ischemic stroke,and whether lncRNA-1810034E14Rik can regulate microglial function after ischemic stroke.In addition,we further explored the molecular mechanism of lncRNA-1810034E14Rik regulating microglial function.Methods:In vitro,primary microglia were extracted from the cortex of C57BL/6 suckling mice,and RNA was extracted after OGD for microarray analysis of lncRNAs.The differentially expressed lncRNAs were screened out and bioinformatics analysis was carried out to select the target lncRNA-1810034E4Rik.Microglia were transfected with a lentivirus that overexpress lncRNA-1810034E14Rik 24 hours before OGD.The secretion of inflammatory factors was detected by ELISA,the activation of microglia was observed by immunofluorescence,the migration ability of microglia was detected by scratch test,and the phagocytic function was detected by fluorescent microspheres.At the same time,microglia and neurons were co-cultured in vitro.CCK-8 and LDH release analysis were used to detect the survival status of neurons.In vivo,we established a model of middle cerebral artery occlusion(MCAO)in C57BL/6 mice.The expression of lncRNA-1810034E4Rik in infarcted cortex was detected by RT-qPCR at different time points after reperfusion.A lentivirus that overexpress lncRNA-1810034E14Rik was injected into the cortex of C57BL/6 mice.MCAO was performed two weeks later and cerebral infarction volume and neurobehavioral function were measured one day later.Immunofluorescence was taken to detect the activation of microglia in ischemic penumbra and ELISA was used to detect the infiltration of inflammatory factors in infarcted cortex.Western blotting was used to detect the NF-kappa B signaling pathway in vivo and in vitro.Results:1)The expression of lncRNA-1810034E14Rik in microglia after OGD was significantly down-regulated(>5 fold change,p<0.05).2)Overexpression of lncRNA-1810034E14Rik significantly reduced the activation of microglia and down-regulated the level of inflammatory cytokine.3)Overexpression of lncRNA-1810034E14Rik had no significant effect on migration and phagocytosis of microglia compared with control group.4)Overexpression of lncRNA-1810034E14Rik could reduce the neurotoxicity of microglia after OGD.5)Overexpression of lncRNA-1810034E14Rik in cortex could improve motor function damage,reduce brain edema and reduce infarcted volume in MCAO mice.6)The NF-kappa B pathway was activated in the infracted cortex and OGD-induced microglia.Overexpression of IncRNA-1810034E14Rik significantly inhibited the phosphorylation of p65.Conclusions:1)LncRNA-1810034E14Rik could reduce infarcted volume and improve neurological function after ischemic stroke;IncRNA-1810034E 14Rik could significantly inhibit neuro-inflammation after ischemic stroke.2)LncRNA-1810034E14Rik could reduce the activation of microglia by inhibiting the NF-kappa B signaling pathway.