| Background: Ischemic stroke(IS)is a common type of multiple cerebrovascular disease.Neuroprotective strategies have attracted extensive attention in the treatment of ischemic stroke patients.Therefore,it is very important to explore the pathophysiology and related mechanism of neurogenesis after cerebral ischemia.It is worth noting that the proliferation of neural stem cells(NSCs)plays an indispensable role in the physiological and pathological process of cerebral ischemia,and has a profound impact on the brain tissue remodeling after ischemic stroke.Objective: This study is the first to investigate whether miR-493-5p,a micro RNA derived from the long non-coding RNA(lncRNA)maternal expressed gene 3(MEG3),influences the proliferation of neural stem cells after ischemic stroke through macrophage migration inhibitory factor(MIF).To investigate the effect of lncRNAEG3 /miR-493-5p/MIF axis on the proliferation of neural stem cells during the occurrence and development of ischemic stroke and its potential mechanism.Methods:1.Endogenous neural stem cells were identified by immunofluorescence staining.We treated neural stem cells with oxygen and glucose deprivation/reoxygenation(oxygen and glucose confinement/reoxygenation,OGD/R)to build an in vitro model.The changes of target lncRNA MEG3 in cell samples were detected by real-time fluorescence quantitativepolymerase chain reaction(RT-qPCR).The proliferation of neural stem cells in normal culture and neural stem cells induced by oxygen and glucose deprivation were evaluated by CCK-8(Cell Counting Kit-8),and then the cell proliferation curve under different conditions was plotted.2.The expression level of miR-493-5p in OGD/R model was detected by RT-qPCR.The possible binding sites of MEG3 and miR-493-5p were predicted on Diana-lncbase v.2.LncRNA MEG3 was knocked down by plasmid RNA transfection technology.Then,the cell transfection was observed with inverted fluorescence microscope and the expression of MEG3 was detected by RT-qPCR to verify the transfection efficiency.The corresponding miR-493-5p mRNA expression levels in the control group and the experimental group were detected by RT-qPCR to verify the correlation between MEG3 and miR-493-5p.3.The levels of MIF mRNA and MIF protein in OGD/R group and blank control group were detected by RT-qPCR and Western blot(WB).The expression of miR-493-5p was interfered by transfection of miR-493-5p mimics and inhibitor plasmid DNA.The transfection efficiency was verified by q RT-PCR,and the change of MIF expression at mRNA level was detected.Western blot was used to further clarify the changes of MIF expression.After the neural stem cells were transfected with miR-493-5p inhibitor,OGD/R induction was performed on the transfected cells and normal cultured neural stem cells,and the changes in the proliferation ability of neural stem cells in the transfected group and the control group were detected by CCK8.4.Neural stem cells were transfected with si-MEG3 and miR-493-5p mimics plasmid,respectively,and then treated with OGD/R.The proliferation level of cells in each group was detected by CCK-8.The expression of proliferation-related protein PCNA was detected by immunofluorescence staining assay.Results:1.NESITN and SOX2,the specific markers of neural stem cells,were co-expressed positively.MEG3 level was significantly up-regulated in neural stem cells induced by OGD/R(P<0.01).We found that the proliferation ability of neural stem cells in the OGD/R group was significantly reduced after 24h(P<0.05).2.The expression of miR-493-5p was up-regulated in the OGD/R cell model(P<0.05).Bioinformatics test found that there might be a binding site between MEG3 and miR-493-5p,and the expression of miR-493-5p decreased after MEG3 was knocked down(P<0.05).3.The experimental results showed that in the experiment of promoting the expression of miR-493-5p,the expression levels of both RNA and protein of MIF were down-regulated(P<0.01).The results were opposite when transfected with miR-493-5p inhibitor in vitro(P<0.05).The proliferation ability of neural stem cells in miR-493-5p inhibitor transfection group was significantly improved to a certain extent(P<0.001).4.When MEG3 was low expressed,the proliferation ability of neural stem cells induced by OGD/R was significantly higher than that of the control group(P<0.001),and the expression of PCNA protein was increased(P<0.01).After transfection with miR-493-5p inhibitor 48 h,the proliferation ability of neural stem cells after OGD/R was increased,which was higher than that of blank control group(P<0.001).The proliferation ability of miR-493-5p mimics was significantly lower than that of blank control group 48 h after transfection(P<0.001).Conclusion1.After OGD/R treatment,neural stem cells promoted MEG3 expression and inhibited cell proliferation.2.MEG3 and miR-493-5p present the same changes in gene regulation and expression,and they are positively correlated.Therefore,we infer that a single primary transcript of the MEG3 promoter regulates the expression of MEG3 and miR-493-5p.3.As a downstream target effector of miR-493-5p,miR-493-5p can directly inhibit the expression of MIF,while overexpression of MIF can reduce the damage of OGD/R on the proliferation activity of neural stem cells.4.In conclusion,inhibiting the expression of lncRNA MEG3 can promote the proliferation of neural stem cells by the miR-493-5p /MIF axis and alleviate cerebral ischemia injury,which contributes to further understanding of the pathophysiological process of ischemic stroke and provides a potential gene target for the effective prevention and/or treatment of ischemic stroke. |
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