Study On The Mechanisms Of MicroRNA-126 Overexpression Improves Neurobehavioral Recovery During Cerebral Ischemia In Mice

Background: Angiogenesis provides adequate oxygen and nutrients for ischemic brain tissue,providing necessary factors and anatomical support for nerve regeneration during chronic phase of ischemic.Thus,angiogenesis is beneficial for improvement of chronic phase of ischemic stroke.Studies demonstrated that microRNA-126-3p modulated angiogenesis through inhibiting PIK3R2 and SPRED1,activating VEGF signaling pathway during embryonic development.However,its function in ischemic stroke is not clear.We hypothesized that microRNA-126 overexpression improved ischemia mice neurobehavior.In this study,we performed angiogenesis model in vitro,immunofluorescence,polymerase chain reaction(real-time PCR),immunoblotting(Western blot),Luciferase Gene Reporter and database search.We performed experiments in human umbilical vein endothelial cells and permanent middle cerebral artery occlusion mice.The aims of the current study are: 1)Whether two chains of microRNA-126,microRNA-126-3p and microRNA-126-5p,have different functions on endothelial cells;2)To determine whether microRNA-126 overexpression improved neurobehavior in permanent middle cerebral artery occlusion mice;3)To explore the mechanism of microRNA-126 in permanent middle cerebral artery occlusion mice improvement.Materials and methods: We carried out Matrigel assay to investigate the effect of microRNA-126-3p and microRNA-126-5p on human umbilical venous endothelial cells tube-formation.We carried out transwell assay to investigate the effect of microRNA-126-3p and microRNA-126-5p on human umbilical venous endothelial cells migration.We carried out Brd U assay to investigate the effect of microRNA-126-3p and microRNA-126-5p on human umbilical venous endothelial cells proliferation.We performed permanent middle cerebral occlusion in mice and injected lentivirus microRNA-126 to the ischemic mice,exploring the microRNA-126 effect.With immunofluorescence,polymerase,real-time PCR,Western blot,Luciferase Gene Reporter and database search,we detected the mechanism of microRNA-126 in ischemic mice.Results: In this study,we detected the function of microRNA-126-3p and microRNA-126-5p in HUVECs.The results demonstrated that microRNA-126-3p had a higher ability in angiogenesis compared with microRNA-126-5p(p<0.05).However,there were no significant distinct in promoting HUVEC migration and proliferation abilities between microRNA-126-3p overexpression group and microRNA-126-5p overexpression group.In consideration of the results above,we injected Lv-microRNA-126 to ischemic mice after permanent middle cerebral artery occlusion mice 1week and Lv-microRNA-126 split to Lv-microRNA-126-3p/5p in mice brain.We found Lv-microRNA-126 improved neural behavior(p<0.05)and reduced atrophy volume(p<0.05)in ischemia mice.Further to detect angiogenesis and neurogenesis,we found that Lv-microRNA-126 groups were superior to Lv-GFP group(p<0.05).AKT and ERK signal pathway which were related to angiogenesis and neurogenesis,were activated in Lv-microRNA-126 treated group(p<0.05).PTPN9,a phosphatase,decreased in Lv-microRNA-126 treated group(p<0.05).We determined PTPN9 as the target of mico RNA-126 with real-time PCR,western blot and bioinformation database.Dual-luciferase gene reporter system showed that PTPN9 was the target of microRNA-126-3p and microRNA-126-5p in mice.We determined mice PTPN9 was the target of microRNA-126-5p first time.Conclusion: In this study,we demonstrated that PTPN9 was a target gene of mice microRNA-126-3p and microRNA-126-5p.It revealed that microRNA-126 could improve stroke motor behavior.Moreover,microRNA-126 overexpression promoted ischemia recovery by directly inhibiting PTPN9,activating AKT and ERK signal pathway and then promoting angiogenesis and neurogenesis.