The Role And Mechanism Of MiR-29b On Cerebral Ischemia/reperfusion Injury In Vitro

Objective: The present study was aimed at the cellular level,to explore the effect of miR-29 b in cerebral ischemia/reperfusion injury and the changes of miR-29 b in apoptosis of neuronal cells.whether miR-29 b inhibitor can against protect cerebral ischemia/reperfusion(I/R)injury in vitro and elucidate its underlying mechanisms.Methods:N2a cells were cultured in vitro and the experimen wast randomly divided into six groups:the control group,OGD / R group,OGD / R+ mimics group,OGD /R+inhibitors group,OGD / R+ mimics NC group,OGD / R+inhibitors NC group.OGD/R(Oxygen–glucose deprivation/reoxgenation)environment was established before miR-29 b mimics or inhibitor transfection.We detected the expression of miR-29 b by RT-q PCR in each group.The cell viability was detected by the Cell Counting Kit 8(CCK8).Apoptosis was detected by Annexin V FITC / PI double staining method.Meanwhile,we also tested Mcl-1?Bcl-2?Caspase-3 proteins using western blotting assay.we validated the interaction between miR-29 b and Mcl-1using dual-luciferase assay in N2 a cells.Results:(1)Compared with the control group,miR-29 b was up-regulated in OGD/R treated N2 a cells(p<0.05).(2)We further transfected miR-29 b mimics into hypoxia N2 a cells,The apoptotic rate of N2 a cells increased and the cell viability decreased(P<0.01).The expression of anti-apoptotic protein Bcl-2 and Mcl-1 was down-regulated and the expression of caspase-3 was up-regulated(P <0.01).but we further transfected miR-29 b inhibitors into hypoxia N2 a cells,The apoptotic rate of N2 a cells decreased and the cell viability increased(P<0.05).The expression of anti-apoptotic protein Bcl-2 and Mcl-1 was up-regulated and the expression of caspase-3 was down-regulated(P <0.05).(3)The results of double luciferase assay showed that miR-29 b was picked out with strong regulation of Mcl-1-3'UTR.Conclusion:The neuro-damage impact of miR-29 b and neuro-protective effect of miR-29 b inhibitors on cerebral I/R injury are associated with neural apoptosis through targeting Mcl-1,suggesting a potential therapeutic target for ischemic stroke.