Transcriptome-wide Analysis To Identify The Inflammatory Role Of Long Non-coding RNA-Neat1 In Ischemic Stroke In Mice

Background:Identifying the long non-coding RNA(lncRNA)involved in neuroinflammation after cerebral ischemic stroke promotes discovering potential therapeutic targets.Nevertheless,the regulatory roles of lncRNAs and their underlying mechanism in ischemic stroke are still largely undefined.Methods:In bioinformatics analysis,we downloaded the raw data of whole-genome sequencing(GSE112348)of C57BL/6N male mouse focal cerebral ischemia from the GEO dataset,screened differential mRNAs and lncRNAs by transcriptome analysis.Differential genes at various time points were intersected and then subjected to gene ontology(GO)term enrichment,KEGG pathway enrichment analysis,immune cell infiltration deconvolution calculation and co-expression network analysis to identity lncRNA-Neatl,which is a crucial lncRNA activating microglia.The bioinformatics results were preliminarily verified in the cell experiments by culturing mouse BV-2 microglial cell line,administrating the antisense oligonucleotide(ASO)for knockdown of lncRNA-Neatl,followed by oxygen-glucose deprivation(OGD)experiment to observe the morphological changes of the cells and to detect the changes in CCK-8,LDH levels and inflammatory factor secretion.In the animal experiment,we established a mouse middle cerebral artery occlusion(MCAO)model,knocked down the lncRNA-Neatl transcript level in the central nervous system by stereotactic intraventricular injection of ASO before modeling,detected the lncRNA-Neatl expression level in the ischemic cortex by qPCR 24 hours after MCAO,scored the behavior of mice and measured the changes in cerebral infarction volume,brain edema content,and recorded the survival rate.ELISA detected the expression levels of inflammatory factors in the infarcted hemisphere,morphological changes were observed by HE staining and Nissl staining,and the activation ratio of microglia and the amount of apoptosis in the infarcted lesion were observed by immunohistochemistry and fluorescence immunostaining.Finally,we detected the levels of HMGB1 protein,apoptosis-related proteins,and JAK/STAT pathway phosphorylation by Western blotting.Results:Bioinformatic analysis and experiments confirmed that lncRNA-Neatl promotes the activation of microglia and promotes the inflammatory response in the pathological process of ischemic stroke.Knockdown of Neatl significantly inhibited the activation of microglia,remarkably decreased the release of microglial pro-inflammatory cytokines IL-1β,IL-6,and TNF-α,increased the levels of cytokines IL-4 and IL-10,attenuated the expression of apoptotic pathway proteins,and reduced the number of neuronal apoptosis.In addition,knockdown of Neatl can also inhibit JAK/STAT pathway activity and the upregulated expression of HMGB1,which is closely related to inflammation downstream.Conclusions:Neatl plays a pro-inflammatory role in activating microglia,and Neatl knockdown may inhibit neuronal apoptosis and improve neurological function in ischemic stroke.This study contributes to a better understanding of the pathologies of ischemic stroke and the development of novel therapeutic options for this disease.