The Effect And Mechanism Of Long Non-coding RNA PEG11as In Cerebral Ischemia Reperfusion Injury

BackgroundIschemic stroke has the characteristics of high morbidity,high disability,high mortality and high recurrence rate,and it has become a major disease that seriously threatens human health.At present,recombinant tissue plasminogen activator(rt-PA)and mechanical clot removal have been used clinically to treat acute ischemic stroke.However,due to the short therapeutic window(normally 3-4.5h after onset)and the complicated pathological mechanism,the clinical treatment of ischemic stroke has not yet made breakthrough progress.A large number of studies have reported that neuronal apoptosis and necrosis induced by ischemic/reperfusion(I/R)have been considered as one of the main pathophysiological mechanisms leading to brain damage.Therefore,focusing on the pathogenesis of inducing neuronal apoptosis,we might find the key molecules in the gene regulatory network,which is expected to provide new research approch for the prevention and treatment of acute ischemic stroke.Long non-codingRNAs(lncRNAs)is a type ofRNA with a length of more than200 nucleotides in eukaryotes.LncRNAs widely exist in tissues and cells,which regulate gene expression at the level of epigenetic,transcription,post-transcriptional and play an important role in many biology process.The abnormal expression of lncRNAs are closely related to the occurrence of cerebral ischemia reperfusion injury.Therefore,searching for lncRNAs,which are related to cerebral ischemia-reperfusion injury,is the important approch that explores their potential molecular mechanism,leading to the new targets for the treatment of ischemic stroke.ObjectiveTo obtain lncRNAs that is related to the pathological process of cerebral ischemia-reperfusion injury byRNA sequencing(RNA-Seq)technology,and further to explore the role and its molecular mechanism of the target lncRNA in cerebral ischemia-reperfusion,which might provide a theoretical basis for the prevention and treatment of ischemic stroke.MethodsPart 1.RNA-Seq technology was used to detect lncRNA expression profile in mouse cerebral I/R injury.Twelve C57BL/6 mice were randomly divided into the sham and cerebral I/R groups(n=6).The suture method was used to construct the model of middle cerebral artery occlusion(MCAO)in mice.After ischemic 1h and reperfusion 6h(I1h/R6h),brain tissue samples were collected to extract totalRNA,the expression prifile was detected byRNA-Seq sequencing.We screened the lncRNAs related to cerebral I/R and established the lncRNA differential expression database according to the standard of |log2(Fold change)|>1,P<0.05,and then the analysis of Gene Ontology(GO)and the Kyoto Encyclopedia of Genes and Genomes(KEGG)for the differentially expressed genes using bioinformatics methods.Quantitative real time polymerase chain reaction(q RT-PCR)was used to validate the genes selected fromRNA-Seq.Among them,the up-regulated lncRNA PEG11 as was identified as the target lncRNA involved in cerebral I/R.Part 2.The effect of lncRNA PEG11 as on cerebral I/R-induced neuronal apoptosis.(1)The effect of lncRNA PEG11 as on cerebral I/R-induced neuronal apoptosis in mice: C57BL6 mice were intraventricular injection with the interfering lentivirus of lncRNA PEG11as(LV-ShRNAPEG11as)or its negative lentivirus(LV-ShRNA NC)for7days,and then subjected to MCAO/R.After 24 h reperfusion,TTC staining,neurological score,TUNEL staining and western blot were used to evaluate the effect of lncRNA PEG11 as on infarct volume,neurological deficit,neuronal apoptosis and the expression of apoptotic related genes in brain tissue.(2)The effect of lncRNA PEG11 as on OGD/R-induced apoptosis in N2 a cells: N2 a cells were transfected with lncRNA PEG11 as interfering plasmid(ShRNA-PEG11as)or its negative plasmid(ShRNA-NC)for 48 h,and then exposed to OGD 3h/R 24 h.TUNEL staining and western blot were used to evaluate neuronal apoptosis and the level of the apoptotic related genes.Part 3.The mechanism of lncRNA PEG11 as on regulating neuronal apoptosis in cerebral I/R injury:RNA-FISH was used to determine the subcellular location of lncRNA PEG11as;furthermore,the bioinformatics analysis was used to predict that lncRNA PEG11 as could be used as a competitive endogenousRNA(ceRNA)in cerebral I/R damage.The analysis ofRNA-binding protein immunoprecipitation(RIP),immunofluorescence,q RT-PCR and western blot was used to detect ceRNA interaction of lncRNA PEG11as/mi R-342-5p/Pfn1.TUNEL staining,western blot,hoechst 33258 staining and the activity of caspase 3 enzyme were used to investigate the role of mi R-342-5p and Pfn1 in OGD/R-induced N2 a cell apoptosis.ResultsPart 1.The differential expression of lncRNA involved in mice cerebral I/R.RNA-Seq sequencing results showed that compared with the sham group,a total of 254 lncRNAs were differentially expressed in the cerebral I/R group.Among them,249 lncRNAs were up-regulated and 5 lncRNAs were down-regulated.The q RT-PCR was used to verified the expression of 9 lncRNAs predicted byRNA-Seq,and lncRNA PEG11 as was identified to be the most significantly expression lncRNA.LncRNA PEG11 as expression was up-regulated,and associated with the time-dependent changes in I/R and OGD/R.At I1h/R6 h,the expression of lncRNA PEG11 as was the highest(P<0.01),and then decreased with the extension of reperfusion time.At I1h/R48 h,the expression of lncRNA PEG11 as was returned to normal,and there was no statistically significant difference compared with sham or control group(P >0.05).GO and KEGG pathway analyses identified significantly enriched pathways in cytokines-cytokine receptor signaling pathway,NF-kappa B signaling pathway,MAPK signaling pathway and apoptosis signalling pathway.Part 2.The effect of lncRNA PEG11 as on cerebral I/R-induced neuron apoptosis.Knockdown of lncRNA PEG11 as reduced the neurological deficit score,decreased cerebral infarct volume,suppressed the expression of apoptosis-related protein cleaved caspase 3 and inhibited neuronal apoptosis in mice cerebral I/R.Consistent with the in vivo findings,lncRNA PEG11 as inhibition also significantly reduced the number of apoptotic positive cells and attenuated the rate of apoptosis,decreased the protein expression of cleaved caspase3,and thereby inhibit OGD/R-induced apoptosis in N2 a cells.Part 3.lncRNA PEG11 as regulates neuronal apoptosis associated with the lncRNA PEG11as/mi R-342-5p/Pfn1 pathway.FISH subcellular localization revealed that lncRNA PEG11 as was mainly located in the cytoplasm.Bioinformatics predicted that lncRNA PEG11 as could act as a ceRNA for mi R-342-5p to affecting Pfn1 protein expression and participate in the apoptotic process in cerebral I/R-induced.The q RT-PCR results showed mi R-342-5p was downregulated in cerebral I/R and OGD/R.Knockdown of lncRNA PEG11 as significantly upregulated mi R-342-5p expression,which was negatively negatively regulated by lncRNA PEG11 as.RIP assays revealed that lncRNA PEG11 as bound to mi R-342-5p directlly.mi R-342-5p mimic decreased the rate of apoptosis of N2 a cells,surpressed the level of cleaved caspase 3 protein and inhibited OGD/R-induced apoptosis,while effects were reversed after mi R-342-5p inhibitor treatment.Co-transfection of mi R-342-5p inhibitor and shRNA-lncRNA PEG11 as attenuated the pro-apoptotic effect of mi R-342-5p inhibitor.Bioinformatics predicts that Pfn1 was a target gene of mi R-342-5p.Pfn1 protein expression is significantly upregulated in cerebral I/R and OGD/R.Further investigations demonstrated that Pfn1 expression level was negatively correlated with mi R-342-5p and positively correlated with lncRNA PEG11 as.Silencing Pfn1 led to the attenuation in the number of hoechst apoptosis-positive cells and decrease in caspase 3 enzyme activity,ultimately,apoptosis was inhibited.ConclusionIn total,254 lncRNAs were identified in cerebral I/R,of which lncRNA PEG11 as was significantly up-regulated.LncRNA PEG11 as knockdown could reduce cerebral I/R-induced neuronal apoptosis.Mechanistic study further revealed that lncRNA PEG11 as functions as a ceRNA for mi R-342-5p and regulates the Pfn1 expression thus promoting cerebral I/R-induced apoptosis.This study provides a new theoretical basis for lncRNA to participate in the pathological processes of ischemic stroke,and provides a novel intervention target for the clinical prevention and treatment of ischemic stroke.