Protective Mechanisms Of MiR-27a Against Oxygen-glucose Deprivation-induced Injuries In Hippocampal Neurons

Objective:During the perinatal stage,ischemia and hypoxia are the main causes of infant(hypoxic-ischemic encephalopathy,HIE).The aim of the study was to axplore the effect of microRNA-27a(miR-27a)against ischemia and hypoxia in rat hippocampal neurons and to investigate the protective mechanisms of miR-27 a.The present study could supply basic for miR-27 a as a potential therapeutic target in treatment of hypoxic-ischemic encephalopathy.Methods:(1)Rat primary hippocampal neuron cultures were prepared from E18-E19 Sprague-Dawley(SD)rat embryos.The cell model of ischemia and hypoxia in vitro(oxygen-glucose deprivation model)was established.MTT(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide)assay was used to measure the cell viability in the hippocampal neurons after treated with oxygen-glucose deprivation(OGD).(2)qRT-PCR(Quantitative Real-time PCR)was used to detect the expression of miR-27 a in the hippocampal neurons exposed to OGD/reoxygenation and neurons without treating.(3)Transfecting with miR-27 a mimic to overexpress mi R-27 a in the rat hippocampal neurons.qRT-PCR was used to detect the expression of miR-27 a in the hippocampal neurons after transfection.(4)MTT assay was used to analyze cell viability of hippocampal neurons transfected with miR-27 a mimic.(5)Flow cytometry(FCM)and TUNEL analysis were used to measure the effect of overexpression of miR-27 a on the cell apoptosis in the hippocampal neurons induced by OGD/reoxygenation.(6)Online softwear(http://www.targetscan.org/)was used to analyze the possible target gene of miR-27 a.(7)The luciferase reporter gene system was used to test the FOXO1 was the target gene of miR-27 a.(8)Western blot was used to detect the effect of overexpression of mi R-27 a on the expression of FOXO1 in hippocampal neurons.(9)Recombined expression vector pcDNA3.1-FOXO1 was constructed,and pcDNA3.1-FOXO1 with mi R-27 a mimic was co-transfected into hippocampal neurons.Rescue experiments were performed to further ensure that the role of mi R-27 a for protecting neurons in OGD-treated neuron was due to regulating target gene FOXO1.(10)Western blot was used to investigate the effect of overexpression of miR-27 a on the protein(pro-caspase-3 and cleaved caspase-3)involved in apoptosis in hippocampal neurons following OGD/reoxygenation treatment.Results:(1)The rat primary hippocampal neurons were cultured successfully.The results of MTT assay showed that during 6–24 hours of reoxygenation post-OGD,neuronal cell viability decreased compared with normal control(normoxia)(P< 0.05).Neuronal cell viability exposed to OGD/reoxygenation for 12 and 24 hours was significantly decreased compared with normoxia control(P < 0.01).(2)The results of qRT-PCR showed that the relative expression levels of mi R-27 a gradually decreased compared with control(normoxia)(P < 0.05).(3)The results of qRT-PCR showed that the relative expression levels of miR-27 a significantly increased in the hippocampal neurons after transfection with miR-27 a compared with normal control or negative control(mimic control)(P < 0.01).(4)The results of MTT assay showed that miR-27 a overexpression promoted cell viability compared with untransfected control(blank)and mimic control(P < 0.05)in the hippocampal neurons following OGD/reoxygenation.(5)The results of FCM and TUNEL analysis showed that the percentage of apoptotic neurons transfected with miR-27 a mimic was significantly less than in the untransfected control(blank)and mimic control(P<0.01)in the hippocampal neurons following OGD/reoxygenation.(6)Online softwear(http://www.targetscan.org/)was used to analyze the possible target gene of miR-27 a.(7)The results of test of the relative luciferase activity showed that luciferase activity was significantly decreased by miR-27 a when the wild type FOXO1 3'UTR was present(P<0.01).(8)The results of Western blot showed that the expression of FOXO1 was significantly reduced in hippocampal neurons transfected with miR-27 a mimic as compared to untransfected control(ctrl)and mimic control(ctrl)(p<0.01).(9)The results of rescue experiments showed that overexpression of FOXO1 could alleviate the protective effect of overexpression of miR-27 a in hippocampal neurons by MTT and flow cytometry analysis.(10)The results of Western blot showed that the expression of cleaved Caspase 3 significantly increased in neurons with OGD/reoxygenation treatment as compared to normoxia(p<0.01)while the expression of pro-Caspase 3 was slightly decreased.Meanwhile,miR-27 a inhibited the expression of cleaved Caspase 3 after OGD/reoxygenation treatment as compared to untransfected control and mimic control(p<0.05).Conclusion:(1)The cell model of ischemia and hypoxia in vitro(OGD model)was established successfully.(2)The expression of mi R-27 a was depressed in the OGD model of hippocampal neurons.Overexpression of miR-27 a could promote cell viability and reduce the cell apoptosis in the hippocampal neurons following OGD/reoxygenation treatment.(3)FOXO1 was the target gene of miR-27 a.Mi R-27 a could depresse the expression of FOXO1 in the hippocampal neurons.Overexpression of FOXO1 could alleviate the protective effect of overexpression of miR-27 a in hippocampal neurons.(4)MiR-27 a may play an important role in the neuroprotection against OGD/reoxygenation mediated by modulation of apoptosis-related gene Caspase 3 expression and FOXO1.