Mmu-miR-124-3p Inhibits Neuronal Apoptosis And Axonal Damage Induced By Oxygen And Glucose Deprivation

BackgroundIschemic stroke is a major cause of death and disability worldwide,and is characteristics of high morbidity,high disability,high mortality,etc.Clinically,the most effective treatment is administration of clot-dissolving drugs or intravascular interventional thrombectomy,to dredge the infarct blood vessel and restore blood oxygen supply to brain tissue.However,dredging the infarct blood vessel may cause the second damage in the ischemic area,named Cerebral ischemia reperfusion(CIR).There are many complicative mechanisms of CIR.Latest research found that after the polarization,microglia drive to M2-microglia which could reduce the cerebral infarction area when M2-microglia was injected via tail vein in CIR model mice.This phenomenon prompt that M2-microglia play a critical role in preventing neurons death and modulating neurobehavioral recovery.In response to brain injury,M2-microglial secreted exosomes act as the cellular communicational components.Exosomes mainly transmit bioinformation through microRNA(miRNA).The miRNA is a single-stranded structure of 20-24 nucleotides in length,which binds to the 3'UTR region of its target gene mRNA,and inhibits the expression of the target gene by inhibiting translation and promoting the degradation of the target gene.And each miRNA has multiple target genes,and modulating the diversity of downstreams can be compiled into a regulatory network.Based on the above,we consider whether miRNAs in exosomes released from M2-microglia have protective effects on neuron,which downstream genes may be the culprit inducing the CIR neuronal apoptosis or be the protector regenerating of neuron axons.This thesis has conducted in-depth research on these issuesAimThis study aims to releave M2 microglia-derived exosomes attenuated ischemic brain injury and promoted the regeneration of neuronal axons via exosomal mmu-miR-124-3p and its downstream target ROCK2 and RNF38.The main purpose is to provide the new mechanism of CIR especially through the cellular communication,and also to provide the new targets and strategies of the prevention and treatment of CIRMethodsFollowing the RNA sequencing and bioinformatics methods,axon regeneration tendentious selection gene analyzed in different brain tissue samples of cerebral,ischemia-reperfusion and sham operation group.LPS and IL-4 were used to construct M1 and M2 type microglia models respectively,extract and identify exosomes.Then RNA was extracted from exosomes by using high-throughput qPCR array to analyze,which screen out the differentially expressed miRNA between M1 and M2 type microglia exosomes.HT-22 and PC12 cells were used to construct OGD models,compared with the bioinformatics analysis and experimental data,found out the expression differences of miRNA and mRNA.Through the above,mmu-miR-124-3p was selected as the upstream of this study,RNF38 and ROCK2 as the downstream target.Bioinformatics databases and luciferase assay experiments was used to determine ROCK2 and RNF38 as the targets of mmu-miR-124-3p.For neuronal apoptosis detection,the OGD model constructed by HT-22 cells which was transfected with mmu-miR-124-3p mimics/inhibitor,using CCK-8 kit,LDH kit,and flow cytometry to detect cell survival rate,LDH viability and apoptosis rate,respectively.For neuronal axon regeneration detection,the OGD model constructed by PC 12 cells which was transfected with rno-miR-124-3p mimics/inhibitor,using Western Blotting and immunofluorescence to observe the neuronal axon markers or regeneration related proteins.For neuronal axon regeneration detection,the OGD model constructed by PC 12 cells which was transfected with rno-miR-124-3p mimics/inhibitor.Result(1)After animal modeling,cortical tissue sequencing analysis revealed that there were around 200 miRNAs and mRNAs differentially expressed in the combination of TC RNA-seq sequencing and AGO CLIP sequencing.The screening results showed that the level of ROCK mRNA was at the discrete edge,TC RNA-Seq sequencing results showed that ROCK2 mRNA was highly expression in the ischemic group and low in the control group;AGO sequencing results showed that the ROCK2 mRNA level in the ischemic group was more in the non-binding than the binding and the control group was the opposite,suggesting that the results are supplementary for TC RNA-seq analysis.The miRNA screening result is that mmu-miR-124-3p is showing highly expression in the ischemic group and low in the control group during TC RNA-seq sequencing;AGO binding capacity sequencing results show that most of the mmu-miR-124-3p is not bound in the ischemic group while the expression is low,and the control group is reduced due to the binding.Since the unbound expression is high,increasing mmu-miR-124-3p content enhances the inhibitation.(2)The M1 and M2 microglia models were successfully constructed,separating from the normal microglia.Flow cytometry showed that the construction rate of M1 microglia reached 86.7%and the M2 microglia reached 90.3%.Exosomes were extracted and identified morphology by transmission electron microscopy(TEM),the existence of most exosomes was observed to be 100 nM.Then Western Blotting detected characteristic markers of exosome,specific in CD63,Tsg101,CD81 and Hsp70,and cell marker GAPDH not expressed,confirmed to be exosomes.(3)mmu-miR-124-3p is highly expressed in exosomes secreted by M2 microglia,but relatively low in M1 microglia derived exosomes.It was not obviously observed the differential expression of mmu-miR-124-3p between non-modeling and OGD-modeling in HT-22 cells,indicating that mmu-miR-124-3p would not have relatively participation in the pathological phenomenon while the nerve cell HT-22 after OGD modeling.mmu-miR-124-3p externally delivered into nerve cells could be a pharmacological treatment like the chemical drugs,and exosomes act as communicational tools.100 nM of mmu-miR-124-3p stimulation increased the survival rate of HT-22 nerve cells under OGD modeling and reduced the rate of apoptosis.(4)Luciferase assay have identified ROCK2 as the target gene of mmu-miR-124-3p.Then the Western Blotting demonstrated that the M2 exosome-derived mmu-miR-124-3p inhibited the expression of downstream proteins MMP2,MMP9,cleaved Caspase3 and Cleaved Caspase9 by inhibiting the expression of ROCK2,and also againsted neuronal cell apoptosis.(5)mmu-miR-124-3p derived from M2-EXO promotes the release of NREP by inhibiting the expression of RNF38 protein,affecting the axon regeneration-related proteins ?-tubulin-?,NF200,GAP43,and promoting axon regeneration.ConclusionThis study releaved that mmu-miR-124-3 p originated from M2 microglia-derived exosomes protects neurons against ischemia-reperfusion-induced injury through inhibiting ROCK/pMLC/Caspase3.Meanwhile mmu-miR-124-3p originated from M2 microglia-derived exosomes promote regeneration of neuronal axon under the OGD modelling.