The Effect Of Long Non-coding RNA N1LR On The Survival Of Neurons After Ischemic Stroke

Background:Stroke is a typical cerebrovascular disease led to high mortality and severe disability in adults.Ischemic stroke makes up approximately 87%of all stroke cases and has been intensively studied.Although great progress has been made in understanding the delicate mechanism of stroke-induced neuronal damage,there is still a lack of effective drugs to reverse neuronal deficits induced by ischemia.Long noncoding RNAs(IncRNAs)are a set of transcripts that range from hundreds to thousands of bases in size and possess little ability to encode proteins.Increasing evidence suggest that IncRNAs have various biological functions and play important roles in the pathophysiological mechanisms of many diseases.However,stroke-responsive IncRNAs rarely have been reported.We carried out a comprehensive expression profiling analysis of lncRNA in the ischemic penumbral cortex of rats using an ArrayStar Rat LncRNA microarray and gained a subset of IncRNAs differentially expressed in the ischemic penumbra earlier.In the present study,we identified the IncRNA-N1LR as a novel ischemia/reperfusion-induced(I/R-induced)lncRNA from the IncRNA expression profiles and assessed the functions of lncRNA-N1LR by silencing and overexpressing the IncRNA in vitro and in vivo in mice.This study may expand the field of functional biology of lncRNAs and provide a potential therapeutic target for I/R-induced neuronal injury.Methods:In this study,focal cerebral ischemia was induced by middle cerebral artery occlusion(MCAO)method using an intraluminal silicon-coated filament.Meanwhile,to imitate the injury of ischemic stroke in vitro,the mouse neuroblastoma cell line Neuro-2a(N2a)was used to establish the OGD/R model.Then the expression pattern of N1LR was measured in the in vivo and in vitro models using RT-qPCR analysis.We constructed the NILR-mRNA co-expression networks and the functions of mRNAs that directly link to N1LR were found in NCBI RefSeq,and the role of N1LR in ischemic stroke was characterized based on the function of mRNA link to it.5’-RACE and 3’-RACE analyses were performed to indentify the transcriptional initiation and termination sites of N1LR,and the size of N1LR was validated by northern blot analysis.N1LR overexpression and interference models were established by N1LR overexpression adenovirus and RNA interference technology,respectively.And the functions of N1LR were assessed by overexpressing and silencing this IncRNA in vitro and in vivo.The influence of N1LR on the cell viability was assayed using the Cell Counting Kit-8(CCK-8),and the cell cycle distribution and cell apoptosis were evaluated using the FACS verse flow cytometer.RNA-FISH was performed to evaluate the location of N1LR in the ischemic penumbra in response to ischemic injury.A 10-score system was used to determine the neurologic deficits in the MCAO animals twenty-four hours after reperfusion,and then the infarct volume was evaluated through TTC-staining.The TUNEL technique was used for the detection and quantification of apoptosis in the ischemic penumbra.Meanwhile,Nissl staining was performed to determine the neural cell damage in the ischemic penumbra.Mechanistically,the Nckl mRNA levels were detected by RT-qPCR,and the p53 protein expression levels were detected by Western blotting.The level of p53 phosphorylation(Ser15)was analyzed using flow cytometer.Result:The expression level of N1LR in the ischemic penumbra was abruptly increased,reaching its maximum after 0.5 h of MCAO.It was then down-regulated in a time-dependent manner along with the prolonged ischemic time.Meanwhile,the trend of the expression pattern of N1LR in the OGD/R-injured N2a cells presented similar to that observed in the in vivo I/R model.N1LR levels reached their peak after 4 h of reoxygenation and then decreased in a time-dependent manner to 0.28 times the control following 16 h of reoxygenation.N1LR appeared to have no effect on the cell cycle progression of N2a cells at the basal state,but it did lead to a reduction in the G0/G1 population in IncRNA-N1LR-overexpressing N2a cells 16 h post-OGD/R.Thus,N1LR specifically releases N2a cells from the G0/G1 phase cycle arrest induced by OGD/R.Meanwhile,an increasing S-phase population was detected in the lncRNA-NlLR-overexpressing N2a cells 16 h post-OGD/R,which indicated more active proliferation.The results of the CCK-8 assays showed that cell proliferation was slightly inhibited by the knockdown of N1LR and was distinctly enhanced by the overexpression of N1LR in N2a cells 16 h post-OGD/R.Annexin V-conjugated FITC and PI staining were perfomed to evaluate the percentage of apoptotic cells and the results showed that the level of apoptosis induced by OGD/R injury was significantly decreased in lncRNA-N1LR-overexpressing N2a cells but was increased in lncRNA-NlLR-knockdown N2a cells.In C57BL/6 J mice subjected to 2 h of MCAO and 24 h of reperfusion,overexpressing N1LR significantly reduced the infarct volume and neurological deficit,while silencing N1LR resulted in the opposite effect.The results of the TUNEL assay in the ischemic penumbra showed that I/R-induced neuronal apoptosis was significantly reduced by overexpressing N1LR and that it was enhanced by silencing N1LR.Consistent with these results,neural cell loss in the ischemic penumbra was markedly attenuated upon overexpressing N1LR,and aggravated following the knockdown of N1LR.Mechanistically,although the depletion of N1LR slightly up-regulated Nckl mRNA expression in N2a cells at a basal state and 16 h post-OGD/R,the overexpression of N1LR appeared to have no effect on Nckl transcription.So there is not enough evidence to support the regulation of Nckl expression by N1LR.LncRNA-NlLR-overexpressing N2a cells exhibited a significant decrease in p53 phosphorylation on serine 15 16 h post-OGD/R,and IncRNA-NlLR-knockdown N2a cells exhibited the opposite trend.Next,we asked if this decrease was due to changes in p53 total protein levels,and the results showed that there was no difference in p53 total protein levels among different treatments.Conclusion:We identified N1LR as a regulator of I/R injury.Our results revealed that N1LR promoted neuroprotection probably via the inhibition of p53 phosphorylation on serine 15.Thus,we propose that IncRNA-N1LR may serve as a potential target for therapeutic intervention following ischemic brain injury.