The Preliminary Exploration On The Relationship Between MiR-124and Neural Stem Cells Proliferation And Differentiation In The Subventricular Zone After Cerebral Ischemia

Background and Objective:Cerebrovascular disease, with an upward trend morbidity recent years, threatenshumans’s health seriously, brings great burden to families and society. Thoughimprovements have been gained on the therapy of stroke, little do we know about theoutcome of patient, especially in the prognosis of ischemic stroke. Recent studieshave already demonstrated the exiStence of neural Stem cells (NSCs) or neuralprogenitor cells (NPCs) in subventricular zone (SVZ) and subgranular zone (SGZ),no matter in human beings, rodents or non-human primates.This NSCs wereactivated after cerebral ischemia and induced to proliferation and differentiation intofunctional mature neurons, integrated into the inherent neural network, can play to acertain extent partially supplement and repair of nerve injury, However, the limitedproliferation and differentiation capacity of endogenous NSCs was not strong enoughto cover the demand of clinical neurological recovery. Therefore, it is of significantimportant to improve the proliferation and differentiation ability of NSCs to themaximum extent after cerebral ischemia, further studies and a better understanding ofthe molecule regulation mechanism behind the proliferation and differentiation ofadult NSCs may provide a new strategy to solve this problem.MicroRNAs (miRNAs) are a class of small noncoding RNAs which throughpost-transcriptional regulate target genes expression, participate a variety ofbiological processes and play an important physiological role in eukaryote. miR-124is a tissue-specific MicroRNAs which highly express in the brain and recent studiesimplicate that miR-124plays a key role in the proliferation of NSCs However,whether the expression of miR-124changes or it is involved in the proliferation anddifferentiation of NSCs after brain ischemia injury are still remained largely unknownand less reported.In this study,we check the level of miR-124and its target genesexpression in the ischemia mice model’s SVZ at different time points to discover therole of MicroRNAs in the regulate of NSCs proliferation and differentiation after cerebral ischemia.Methods:1. Cerebral ischemia Sprague-Dawle(SD) rats model and experiment groupsSprague-Dawle(SD)rats were randomly divided into sham operation group andcerebral ischemia group, ischemia group with intraluminal suture method in rats withmiddle cerebral artery occlusion (middle cerebral artery occlusion, MCAO) modelof cerebral ischemia reperfusion, at each time point (1d、3d、7d) were sacrificed, andbrain tissue SVZ specimens to be seized.2. Proliferation of endogenous NSCs in the SVZ after cerebral ischemiaImmunofluorescent staining was used to check the expression of Nestin positivecells in SVZ at different time points in sham group and cerebral ischemia grouprespectively.3. Expression change of miR-124in the SVZ after cerebral ischemiaReal-time PCR was used to examine the miR-124expression changes in the SVZat different time points in sham group and cerebral ischemia group respectively.4. Expression change of miR-124’s target gene Sox9mRNA in the SVZ aftercerebral ischemiaReal-time PCR was used to examine the sox9mRNA expression changes in theSVZ at different time points in sham group and cerebral ischemia group respectively.Results:1. Successful establishment of cerebral ischemia in SD modelSprague-Dawle (SD) rats showed different degrees of the neurological deficitssuch as contralateral hemiplegia, ptosis, walking around in place,tilt to the oppositeside, and raised tail sideways to the opposite side after cerebral ischemia. The brainshowed normal red tissue and pale ischemic region by2,3,5-triphenyl tetrazoliumchloride (TTC) staining.2. Proliferation of endogenous NSCsImmunofluorescence staining results: Compared to sham group, the nestinpositive cells in the SVZ were significantly increased and were up-regulated(3.0±0.13) fold,(4.5±0.24) fold and (6.5±0.29) fold respectively after cerebral ischemia1days,3day and7days, revealing that cerebral ischemia can induce theproliferation of endogenous NSCs in the SVZ.3. Expression change of miR-124and Sox9mRNA in the SVZ after cerebralischemia3.1Expression change of miR-124Real-time PCR outcome: Compared to sham group, the expression levels ofmiR-124were significantly up-regulated（2.12±0.23）fold,(3.56±0.34) fold and(2.85±0.27) fold (P<O．05) respectively after cerebral ischemia1days,3day and7days, suggesting that cerebral ischemia can up-regulated the expression of miR-124.3.2Expression change of Sox9mRNAReal-time PCR outcome: Compared to sham group, the expression of Sox9mRNA were apparently down-regulated (0.61±0.11) fold,(0.13±0.15) fold and(0.19±0.21) fold (P<O．05) respectively after cerebral ischemia1days,3day and7days. These results suggested that cerebral ischemia can down-regulated theexpression of Sox9mRNA.Conclussions:1. Cerebral ischemia injury can induce the proliferation of endogenous NSCs inthe SVZ.2. The expression of miR-124was significantly increased after cerebral ischemiawhereas the expression of its target gene--Sox9mRNA was apparentlydown-regulated which suggests that the miR-124/Sox9signal pathway may play akey role in the regulation of NSCs proliferation in SVZ after cerebral ischemia.