The Neuroprotective Effect And Mechanism Of MiR-181a After Oxygen Glucose Deprivation Reperfusion

Objective:To investigate the role of miR-181 a in oxygen glucose deprivation and reperfusion injury and its possible mechanism of N2 a neuroma cells.At the same time,we further study the effect of miR-181 a on the regulation of the expression level of target protein(Reln)and the Smac-IAPs signaling pathway after N2 a cells oxygen glucose deprivation and reperfusion.Methods: mouse neuroblastoma N2 a cells were selected as the research objects,and N2 A cell OGD model was used to simulate neuronal ischemic injury.They were randomly divided into 5 groups,which were normal control group,oxygen glucose deprivation group for 4 hours and reperfusion group at different time.The cell apoptosis rate and its changes were detected by flow cytometry.The expression level of mi R-181 a was detected by real-time quantitative PCR.The expression level of Reln protein was detected by Western blot.The direct regulation effect of miR-181 a on Reln-3 ’UTR was detected by CO transfection of double luciferase reporter gene.Then,miR-181a-mimics up-regulated the expression of miR-181a。Detection of cell apoptosis rate,miR-181 a expression level and Reln protein level in each group.The expression level of Smac mRNA and XIAP mRNA for 24 hours of oxygen glucose deprivation that was detected by real-time quantitative PCR.The expression of Smac and XIAP protein was detected by Western blot.Results: 1.The changes of apoptosis rate of oxygen glucose deprivation and reperfusion: The cell apoptosis rate was increased after the time of oxygen glucose deprivation prolonged.The apoptosis rate of OGD4h/R0 h group was higher than that of the normal control group,but the difference was not statistically significant(P>0.05).OGD4h/R4 h group apoptosis rate was obviously higher than normal control group(P<0.01,with the prolongation of reperfusion,the apoptosis and reperfusion 12 hour group and 24 hour group cells was significantly increased,compared to the control group,the difference was significantly(P<0.01).2.The expression of miR-181 a was significantly decreased with the prolongation of oxygen glucose deprivation reperfusion time.There was difference in the miR-181 a between OGD group compared with the control group(P<0.01).The expression of miR-181 a in OGD4h/R4 h group significant increased compared with the control group(P<0.01).The OGD4h/R12 h and OGD4h/R24 h group of miR-181 a was increased significantly comparing with the control group(P<0.01).3.miR-181 a overexpression group was compared with the normal control group and the reagents control group,and the apoptosis rate of the cells decreased significantly after 24 hours of oxygen glucose deprivation(P<0.01).4.After overexpression of miR-181 a,the Reln protein decreased significantly.The results of double luciferase reporter gene detection showed that miR-181 a significantly inhibited luciferase activity(fluorescence ratio =0.773).5.The level of Smac in the miR-181 a overexpression group was significantly lower than that in the normal control group and the reagents control group(P<0.01),but the level of XIAP was slightly higher.Conclusion: 1.After the oxygen glucose deprivation reperfusion,the apoptosis rate of N2 a cells was increased.MiR-181 a can reduce the apoptosis of nerve cells and protect the nerve cells.2.In oxygen glucose deprivation reperfusion injury,miR-181 a was directly associated with the Reln gene 3 ’UTR fragment and negatively regulated the level of Reln protein.3.miR-181 a can protect the nerve after oxygen glucose deprivation and reperfusion by mediating the Smac-IAPs signal pathway.