| Background:The reperfusion injury after cerebral ischemia threat to human life and health seriously.Nurr1 is a member of the nuclear receptor 4 family of orphan nuclear receptors.It is decreased in inflammatory responses and leads to neuronal death in Parkinson’s disease.Increasing recent evidence suggests important roles for microRNAs(miRNAs)in molecular processes of cerebral ischemia pathogenesis,which involve fast post-transcriptional effects and simultaneous regulation of various target genes.Abnormal expression of Nurr1 has been attributed to various signaling pathways,but little is known about microRNA(miRNA)regulation of Nurr1 in ischemia/reperfusion injury.Objective:In the present study,we therefore sought to determine:(1)How Nurr1 expression and Nurr1-related microRNAs expression change in the brain of MCAO/R rats,an in vivo model;(2)in in vitro studies,what are the post-transcriptional regulatory network of Nurr1 and the specific mechanisms of Nurr1 on microglia activation;(3)in an in vivo study,whether miR-145-5p/Nurr1/TNF-a signal exerts neuron injury function upon cerebral ischemia-reperfusion in rats.Methods:(1)Cell Culture of Primary Microglia and Neurons Experimental.(2)Coculture of Microglia and Neurons and OGD/R Treatment.(3)Construction of Middle Cerebral Artery Occlusion and Reperfusion(MCAO/R)Model for in Vivo Experiments,Neurological Outcome Assays and Infarct Volume Analysis.(4)RNA Extraction,Reverse Transcription and RT-qPCR.(5)miR-145-5p mimic and anti-miR-145-5p injection via intracerebro-ventricular infusion.(6)Cloning of Nurr1 3’UTR and Dual Luciferase Reporter Assay.(7)Cell Viability Analysis by MTS.(8)Chromatin Immunoprecipitation(ChIP)Assay.(9)Western blot and Immunofluorescence Staining.Results:(1)Construction of Middle Cerebral Artery Occlusion and Reperfusion(MCAO/R)Model for in vivo experiments,to detect Nurr1 expression at 2,6,12,24 and 48 h after MCAO/R.Both mRNA and protein levels of Nurr1 decreased from the start of ischemic stroke,reached a minimum at 12 h,and then increased until 48 h.Rats in the sham group were used as negative control.(2)miRNA expression was measured in our miRNA profiling analysis of rat cortex samples at 2,6,12,24 and 48 h after MCAO/R.Eleven miRNAs,including miR-145-5p,miR-34c-5p,miR-365-3p,miR-214-3p,miR-151,miR-27 a,miR-153-5p,miR-365-3p,miR-33-5p,miR-217-5p and miR-129-5p,were differentially and significantly expressed.Among them,4 putative relevant miRNAs were screened by bioinformatic analysis of databases to identify miRNAs that regulate Nurr1 directly through its 3’UTR,including miR-145-5p,miR-365-3p,miR-34c-3p and miR-217-5p.Mutations of the miR-145-5p and Nurr1 recognition sites in the 3’UTR abolished their interaction.Results show that miR-145-5p is targeted for Nurr1.(3)miR-145-5p expression was assayed in microglia at 0.5h,1.0h,2.0h,6h and 12 h post OGD/R.miR-145-5p expression increased from the onset of OGD/R,peaked at 2 h,and then progressively decreased until 12 hin microglia.However,no significant changes of miR-145-5p expression were observed at different time points of OGD/R in primary neurons.Nurr1 mRNA levels were reduced from the onset of OGD/R and peaked at 2 h in microglia.miR-145-5p Regulates Endogenous Nurr1 Levels in Microglia.These experiments suggest that enhanced levels of miR-145-5p inhibit neuronal viability by regulating expression of Nurr1 and TNF-a in microglia after OGD/R.(4)TNF-a expression was assayed in microglia at 0.5h,1.0h,2.0h,6h and 12 h post OGD/R.Results of qRT-PCR analysis showed that TNF-a mRNA expression increased and peaked at 2 h of OGD/R in microglia which is opposite to Nurr1 mRNA levels,but not in primary neurons.Upon treatment with the Nurr1 activation plasmid,overexpression of Nurr1 significantly attenuated TNF-a mRNA and protein expression.Conversely,an increase in TNF-a expression was observed after treatment with Nurr1-siRNA after OGD/R 2 h in microglia.Results of further in vivo study revealed that anti-miR-145-5p administration brought about increasing expression of Nurr1 and reduction of infarct volume in acute cerebral ischemia.By chromatin immunoprecipitation(ChIP)assay,Nurr1 plasmid transfection increased Nurr1 occupancy at the TNF-a promoter,especially when TNF-a expression reached a high level after OGD/R 2 h.(5)We next administered the miR-145-5p mimic and anti-miR-145-5p in vivo via ICV injection into ischemic rats immediately after MCAO.Mean infarct volumes were measured at 12 h and 24 h post-MCAO/R.Administration of anti-miR-145-5p reduced infarct volume by 24.05% at12 h,whereas administration of the miR-145-5p mimic increased infarct volume by 11.05%.Results of qRT-PCR assay revealed that administration of anti-miR-145-5p significantly increased Nurr1 mRNA expression and reduced TNF-a mRNA expression at 12 h post-MCAO/R but not at 24 h post-MCAO/R.Nurr1 expression increased significantly upon anti-miR-145-5p treatment versece miR-145-5p mimic treatment in active microglia in peri-infarct areas post 12 h of MCAO/R using immunofluo-rescence staining.(6)Significant functional deficits were observed in the mNSS for animals subjected to mimic miR-145-5p administration compared to antimiR-145-5p animals at both 7d and 14 d after injury.For foot fault tests,the percentages of front left,hind left,total left,and total foot faults at 7d and14 d after injury after anti-miR-145-5p was significantly lower,compared to that of null and mimic miR-145-5p animals.miR-145-5p Interruption Facilitates Neurological Outcome of Rats Post MCAO/R.Conclusions:I/R-induced miR-145-5p overexpression suppresses Nurr1 protein expression and attenuates Nurr1 transrepression of the TNF-a promoter in microglia(OGD/R 2h),which then causes TNF-a-related neuronal injury.Additional in vivo studies have shown that administration of anti-miR-145-5p could increase Nurr1 expression and reduce subsequent infarct volume in acute cerebral ischemia(MCAO/R 12h).It may be an effective therapeutic strategy of reducing neuronal injury following MCAO/R in rats by blocking abnormal activation of miR-145-5p/Nurr1/TNF-a axis signaling in the acute phase. |
