MiR-1247-3p Mediates Apoptosis Of Cerebral Neurons By Targeting Caspase-2 In Stroke

Objective To explore the effect of miR-1247-3p on neuronal apoptosis in cerebral ischemia-reperfusion injury(IS/R),and elucidate the possible molecular mechanisms of miR-1247-3p regulating neuron apoptosis.Methods In this study,a mouse cerebral IS/R injury model of middle cerebral artery occlusion and reperfusion(MCAO)model for ischemia 1 h and reperfusion 0 h,6 h,12 h,and 24 h and oxygen-glucose deprivation/reoxygenation(OGD/R)model for oxygen glucose deprivation 3h and reoxygenation 0 h,6 h,12 h and 24 h were used to mimic the ischemia reperfusion injury in vivo and vitro,and the miR-1247-3p expression were detected.Intracerebroventricular injection LV-miR-1247-3p lentivirus 7 days and transfection N2 a cell with 25 nM miR-1247-3p mimic 48 h before constructing ischemia reperfusion model to observe the neurons apoptosis after overexpression miR-1247-3p.20 ?M Caspase-2 specific inhibitor Z-VDVAD-FMK was used to treat N2 a cells to determine whether the mechanism was related to the regulation of caspase-2 expression.According to this,the MRTF-A overexpression plasmid was transfected into N2 a cells,and the effect of MRTF-A overexpression on miR-1247-3p regulating neurons apoptosis was detected.In this study,the brain impairment were analyzed by Longa’s five score method and TTC staining.The apoptosis of neurons was measured by TUNEL staining and the level of cleaved caspase-3 protein expression.miR-1247-3p and miR-1247 RNA expression were detected by RT-PCR,MRTF-A,cleaved caspase-3 and caspase-2 protein level were detected by western blot.Reasults In the model of cerebral ischemia reperfusion,we detected that miR-1247-3p levels was downregulated and a significant decrease after reperfusion 24 h(P < 0.01).And the expression of miR-1247-3p was also down-regulated and was the lowest after reperfusion 24 h(P < 0.001)in N2 a cell induced by OGD/R.After overexpression miR-1247-3p in vivo,the average behavior scores(P < 0.05),the area of cerebral infarction(P < 0.01)and the apoptosis ratio were decreased(P < 0.001)of the LV-miR-1247-3p+IS 1h/R 24 h group compared with the LV-NEG +IS 1h/R 24 h group.The N2 a cell were transfected with miR-1247-3p mimic before OGD/R treatment,and the apoptosis ratio of miR-1247-3p mimic + OGD 3h/R 24 h group was also decreased compared with NEG-mimic + OGD 3 h/R 24 h group(P < 0.001),suggesting that miR-1247-3p may inhibit the cerebral ischemia reperfusion injury induced neurons apoptosis.In order to verify the mechanism of miR-1247-3p,we found that Caspase-2 may be a target of miR-1247-3p predicted by the TargetScanVert.The results showed that Caspase-2 protein levels were up-regulated in the in vitro and in vivo models of cerebral ischemia-reperfusion injury(P < 0.001),while overexpression of miR-1247-3p could reduce caspase-2 protein levels(P < 0.01).To further verify the relationship between caspase-2 and apoptosis,we treated N2 a cells with 20 ?M caspase-2 inhibitor Z-VDVAD-FMK and found that Z-VDVAD-FMK reduced the rate of N2 a cell apoptosis in the OGD/R model(P < 0.001)significantly compared with OGD/R group.And Z-VDVAD-FMK reduced the apoptosis ratio of N2 a cells induced by miR-1247-3p inhibitor(P < 0.05).Bioinformatics analysis revealed that there is a CArG box in the upstream promoter region of miR-1247-3p,and we hypothesized that MRTF-A may regulate the expression of miR-1247-3p.The results of RT-PCR showed that the mRNA level of miR-1247-3p was up-regulated after overexpression MRTF-A level(P < 0.001),and MRTF-A overexpression also reversed the miR-1247-3p down-regulation induced by OGD 3h/R24h(P < 0.05).And the level of miR-1247 was also up-regulated after overexpression MRTF-A(P < 0.001),while the level of miR-1247 was decreased in the OGD/R model(P < 0.05),MRTF-A overexpression could also reverse the level of miR-1247 in the OGD/R model(P < 0.01).TUNEL staining and cleaved caspase-3 protein levels also showed that overexpression MRTF-A could reduce apoptosis in the N2 a OGD/R model(P < 0.001),whereas the protective effect of MRTF-A on apoptosis in the OGD/R model was blocked after transfection with miR-1247-3p inhibitor(P < 0.001).Conclusion According to the above results,we concluded that miR-1247-3p may protect neurons from cell apoptosis during brain stroke.And this process may be regulated through MRTF-A-miR-1247-3p-caspase-2 signaling pathway.