MiR-124 Regulates Neural Stem Cells Proliferation In Subventricular Zone Of Adult Rats After Traumatic Brain Injury

BackgroundTraumatic brain injury(TBI)is a major health problem with high incidence,disability and mortality.The mortality and disability of TBI are higher than other kinds of trauma in both daily life and warfare,which have caused great burden to the patients,their families and the whole society.It is estimated that more than 50 million TBI accidents happen around the world every year,with an overall cost of nearly$400 billion which accounts for one third of the global disease burden.TBI is highly heterogeneous due to the diverse features of external forces,mechanisms of impacts,pathologies and severities.The primary injury caused by direct external force can damage the structure and function of brain immediately,resulting in contusion and laceration of brain and diffuse axonal injury,which are often complicated with secondary brain damages such as intracranial hemorrhage and hematoma formation.Additionally,intracranial hematoma may aggravate the severity of brain edema,causing rapid increase of intracranial pressure,which further leads to the dysfunction of the control center of respiratory and circulatory system.In addition,primary brain injury can induce a series of secondary neurochemical changes and neurometabolic abnormalities,including excitotoxicity,mitochondrial dysfunction,metabolic disorders,neuroinflammation,oxidative stress,brain edema and dysfunction of blood-brain-barrier,which are the complex causes of brain dysfunction.For decades,countless explorations have been carried out to improve the outcome of TBI while the prognosis of severe TBI is still unfavourable.More drugs or therapeutic strategies with promising application prospects are needed to be explored in future researches.In adult mammals,there are two neurogenic regions in which neural stem cells(NSCs)reside,ie.the subventricular zone(SVZ)near the lateral ventricle,and the subgranular zone(SGZ)in the dentate gyrus of the hippocampus.Adult NSCs in SVZ region are also called type B cells.The basal side of type B cell extends to the blood vessels,and its cilia threads through the ependymal cell layer to contact with the cerebrospinal fluid(CSF)in the ventricle.Type B cells can produce transient proliferative progenitor cells(type C cells)which can divide several times before transforming into neuroblasts(type A cells).Neuroblasts can enter the rostral migratory stream and migrate to the olfactory bulb where they migrate radially and differentiate into various subtypes of interneurons.It has been confirmed that the neurogenic potential of SVZ can improve significantly after cerebral infarction,suggesting that SVZ NSCs play a role in the restoration of brain injury.Therefore,it will be helpful to explore the mechanism of SVZ NSCs participate in brain injury restoration and to make full use of their features in brain regeneration and restoration to improve the prognosis of TBI.MiRNAs are endogenous non-coding RNAs with a length of 18～22 nucleotides.By binding to the 3’untranslated region of the m RNA,miRNA induces m RNA degradation or post transcriptional translation inhibition,thus regulating the expression of genes at the post transcriptional level.The expression of miRNA is tissue-specific and time-dependent.Under different developmental phases,the expression and distribution of miRNAs in different tissues are different,and their functions may also be diverse.MiRNAs are not only involved in the development of nervous system and adult neurogenesis,but also play important roles in brain injury restoration,and are promising drugs for the treatment of brain injury.MiR-124 is mainly expressed in the central nervous system and is a brain specific miRNA.The expression of miR-124 in the central nervous system is more than 100 times higher than in other tissues,accounting for 25%～48%of the total brain miRNAs.The expression level of miR-124 increases gradually when the neural stem cells differentiate into mature neurons.The expression of miR-124 reaches the peak at the time the new neuron matures,and then maintains unchanged.Transfection of miR-124 into Hela cell down regulates 174 genes expression,and the gene expression pattern of transfected Hela cell is more similar to neuron.Transfection of miR-124 and miR-9 into human fibroblasts can transform them into neurons.In addition,over-expression of miR-124 can promote axonal outgrowth,otherwise inhibit axonal growth.In vivo,miR-124 can directly affect the formation of neurons in adult mammalian neurogenesis.Knockout of endogenous miR-124maintains the NSCs at the state of division precursors,while over-expression of miR-124induces NSCs to differentiate into neurons.In neurodevelopment,miR-124 knockout mice has obvious phenotypic changes in development,such as smaller brain volume,axon defects,and even neural cell death.Since miR-124 plays an important role in regulating the proliferation and differentiation of NSCs,we believe that miR-124 can regulate the functional status of NSCs in SVZ and affect the prognosis of TBI.ObjectiveThe purpose of this study was to explore the effect of miR-124 on the prognosis of TBI and further explore its regulatory role on NSCs in SVZ.Bioinformatic analysis and in vivo experiments were used to explore the profound mechanism of miR-124 function in order to provide new ideas for improving the prognosis of TBI.Methods1)Moderate TBI rat model was established by controlled cortical impact apparatus.The post-TBI expression of miR-124 in SVZ was detected by real-time quantitative PCR.Nestin,GFAP,DCX and Ki67 were detected by immunofluorescence assay to evaluate the count and proliferation status of NSCs in SVZ.2)The efficiency of miR-124 interference with micro-injection of agomir or antagomir was confirmed.The motor function of TBI rats injected with miR-124 analogues or inhibitors was detected by modified neurological severity score(m NSS)and the Rotarod test.Nestin,GFAP,DCX and Ki67 were detected by immunofluorescence assay to evaluate the counts and proliferation status of NSCs in SVZ.3)In order to analyze the biological function of miR-124,online miRNA target prediction databases MiRanda,miRDB and Targetscan were used to predict the possible targets of miR-124.The most reliable targets were selected by combining the results of the three databases,and KEGG pathway enrichment analysis was carried out.In this way,we could infer the function of miR-124 and further find out the pathways related to the proliferation and differentiation of NSCs.4)Western blot(WB)was used to detect the expression changes of key molecules involved in the miR-124 targeted pathways in SVZ tissue of TBI rats after intervention of miR-124 expression in SVZ,so as to verify the functional mechanism of miR-124 regulation.Finally,the upstream molecules of miR-124 targeted pathways were used to verify the regulatory effect of miR-124 on its targeted pathways.Results1)The results of q RT-PCR showed that the expression of miR-124 in SVZ decreased after TBI which maintained a low expression level for a long time.Immunofluorescence results showed that NSCs in SVZ were activated after TBI.The statistics showed that the number of nestin~+/GFAP~+neural progenitor cells,DCX~+neuroblasts and Ki67~+proliferation cells increased gradually after TBI.2)After injection of miR-124 analogues or inhibitors into bilateral cerebral ventricles,the expression level of miR-124 in SVZ changed significantly.Down-regulation of miR-124 improved the motor function of adult TBI rats and enhanced the activation of NSCs in SVZ.Up-regulation of miR-124 did not significantly affect the motor function prognosis or the status of NSCs in SVZ in adult TBI rats.3)887 miR-124 target genes were screened for KEGG pathway analysis.The results showed that the enriched pathways of miR-124 targeted genes were"neurotrophin signaling pathway","prolactin signaling pathway"and so on.Among all predicted targets,Akt3,Ras and PIK3CA were involved in the largest number of pathways.Therefore,Akt3,Ras and PIK3CA were considered as the key targets of miR-124.4)WB results showed that the expressions of BDNF,DCX,Akt3,RAS and PIK3CA in SVZ were up-regulated after TBI.When miR-124 was inhibited by antagomir,the expression of Akt3,RAS and PIK3CA further increased.In addition,down-regulation of miR-124 decreased the expression of phosphorylated molecules downstream of PI3K/Akt3pathway and Ras pathway.After injection of BDNF into TBI rats,the activation of NSCs in SVZ increased.At the same time,the activation of NSCs was further enhanced after combind administration of miR-124 inhibitors.These results implied that the decrease of miR-124 expression could enhance the effect of BDNF on NSCs proliferation.ConclusionsThe expression of miR-124 in SVZ decreases in adult TBI rats and sustains for a long time,and the proliferation of NSCs in SVZ increases.After TBI,lower expression level of miR-124 in SVZ is correlated with better prognosis of the motor function.Meanwhile,down-regulation of miR-124 in SVZ could enhance the activation of NSCs in SVZ.Furthermore,down-regulation of miR-124 could enhance the function of PI3K/Akt3pathway and Ras pathway which are the downstream pathways of BDNF,thereby enhancing the effect of NSCs activation caused by BDNF.