MicroRNA-1906 Ameliorates Ischemic Brain Injury By Targeting Toll-like Receptor 4

Background and PurposeStroke is the leading cause of death and adult disability in China.Among all the stroke patients,87%cases are ischemic.However,the current available therapeutic options against acute ischemic stroke(AIS)are limited.The compelling disease burden on patients and the whole society,and the paucity of therapeutic options underlies the urgency to understand the mechanism of stroke and to find novel potential therapeutic targets.Recent studies have revealed the critical involvement of immune response in AIS.Inflammation plays a paradoxical role in AIS,which is beneficial during the recovery phase but deleterious role during the acute phase.Therefore,immune modulation during the acute phase may be a promising therapeutic approach against aggravating ischemic injuries.Toll-like receptor 4(TLR4)is a key mediator in innate immunity,one of the first line sensor that trigger inflammatory cascades,and a link between innate and specific immunity.Inhibition of TLR4 in preclinical studies was associated with reduced infarct volume and improved functional outcome,suggesting that regulating TLR4 may reduce ischemic injury in the brain by ameliorating post-stroke inflammation.MicroRNAs regulate approximately 30%of genes in the neuronal network.Preclinical studies found that microRNAs alter dramatically in AIS,and play key roles in the progression of AIS.Despite the intensive study of microRNA,specific regulation of TLR4 by microRNAs after cerebral ischemia is not yet fully understood.The current study mainly focused on the regulation of TLR4 in the brain at the microRNA level,and its influence on post-stroke inflammation and AIS.MethodsIn this study,middle cerebral artery occlusion(MCAO)model and oxygen-glucose deprivation(OGD)model were used as AIS models in vivo and in vitro.MicroRNA microarrays and polymerase chain reaction(PCR)were used to screen differentially expressed microRNAs in mouse brain after MCAO.The microRNA that can specifically regulate TLR4 was determined by bioinformatic analysis combined with a luciferase reporter gene system.The temporal and spatial distribution of the microRNA in infarction core and peri-infarct region was detected by PCR and in situ hybridization.The microRNA agomir was administered to primary cultured neurons,astrocytes,and microglia in vitro.TLR4 expression was detected by western blotting(WB)and immunofluorescence(IF).Agomir and antagomir of microRNA were administered via intra-ventricle injection.The expression of TLR4 after MCAO was detected by WB and IF,and the expression of inflammatory factors was detected by PCR.Magnetic resonance imaging(MRI)was used to calculate infarction volume,hematoxylin-eosin(HE)staining,Fleoro-Jade C staining,terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)was used to observe brain tissue injury and modified neurological severity score(mNSS)to evaluate the neurological function of mice.ResultsCombined bioinformatics analysis of TargetScan and miRanda databases,microRNA chip and PCR assays,and luciferase reporter gene system confirmed that microRNA-1906 specifically recognizes 3-terminal untranslated region of TLR4 messenger RNA(mRNA)to inhibit the expression of TLR4.In MCAO models,mismatch between TLR4 expression at the mRNA and protein levels occurred after cerebral ischemia.The mRNA levels gradually increased over time,while TLR4 protein increased at the second hour and returned to baseline levels at the fourth hour.At the same time microRNA-1906 began to accumulate since the second hour,and in situ hybridization showed that microRNA-1906 mainly gathered in the peri-infarction region.Administration of synthetic microRNA-1906 agomir reduced the expression of TLR4 after OGD in cultured primary neurons,astrocytes,and microglia.In vivo experiments also confirmed that microRNA-1906 agomir can inhibit TLR4 protein expression,reduce the expression of pro-inflammatory cytokines,reduce infarct volume and tissue damage,and reduce the severity of neurological deficits,while microRNA-1906 antagomir can increase TLR4 protein expression.ConclusionsThe study identified that miR-1906 is a novel regulator of TLR4,and proved that administration of the miR-1906 agomir to mice can ameliorate brain injury caused by experimental stroke by inhibiting TLR4 expression and post-stroke inflammation in the acute phase.