Study On The Role And Mechanism Of MiR-9a-5p In Hypoxic-ischemic Brain Injury

Objective:Neonatal rats with hypoxic-ischemic brain injury(HIBD)and glycemic oxygen deprivation(OGD)in PC 12 cells were established to detect the expression of miR-9a-5p in pineal gland after HIBD and PC 12 cells after OGD;After miR-9a-5p target gene clock was detected by luciferase assay,mRNA and protein expressions of clock in pineal gland after HIBD and PC 12 cells after OGD were further analyzed;PC 12 cells were transfected with mir-9a-up lentivirus and mir-9a-5p inhibitors to study the regulatory relationship between mir-9a-5p and clock.Lv-rno-mir-9a-1 was injected into the left ventricle to study the effect of mir-9a-5p on long-term behavior changes in newborn rats,and to explore the role and mechanism of mir-9a-5p in hypoxic-ischemic brain injury.Methods:Seven-day-old SD newborn rats were used to establish the HIBD model according to the modified Rice-Vannucci method.They were divided into a sham operation group(Sham group)and a hypoxic-ischemic group(HIBD group)according to the random allocation principle.PC 12 cells were treated with glucose oxygen deprivation to simulate hypoxic-ischemic model.MAP2 immunofluorescence assay for neuron characteristics in PC 12 cells;RT-qPCR was used to detect the expression of miR-9a-5p,Aanat,clock,perl,cryl,bamll after HIBD in rats and oxygen deprivation in PC 12 cells;Expressions of clock,perl,cryl and bamll were detected by Western blot;Changes of Melatonin(MT)in Rat Peripheral Blood and Cell Culture Medium by ELISA;After miR-9a-up lentivirus and miR-9a-5p inhibitor were transfected into PC 12 cells,the above experiments were performed.Morris water maze experiment,mine experiment and new object recognition experiment were performed on 3-month-old SD rats injected with 1v-rno-mir-9a-1.After behavioral test,RT-qPCR was used to detect the expression of mir-9a-5p and clock mRNA in the pineal gland of rats.Results:1.The expression of mir-9a-5p in pineal gland at 5 time points of 0h,12h,24h,36h and 48h after HIBD was higher than that in Sham group at corresponding time points(P<0.05).The expression of Aanat mRNA in the pineal gland at 24h,36h and 48h after HIBD was higher than that in Sham group at the corresponding time point(P<0.05).The secretion of MT at 12h,24h,36h and 48h after HIBD was significantly lower than that of Sham group at the corresponding time point(P<0.05).At 12h,24h,36h and 48h after OGD,the expression of mir-9a-5p in PC 12 cells was higher than that in normal cells(P<0.05),and the MT secretion was lower than that in normal cells(P<0.05).2.The target gene of mir-9a-5p was clock detected by double luciferase assay.The expressions of clock mRNA and protein in the pineal gland at 5 time points after HIBD were lower than those in Sham group at corresponding time points(P<0.05).The expression of clock mRNA in PC 12 cells at 12h,24h,36h and 48h after OGD was lower than that in normal cells(P<0.05),and the expression of clock protein at 5 time points after OGD was lower than that in normal cells(P<0.05).The other clock genes Per1,Cry1,Baml1 mRNA and protein of mammalian circadian clock center were down-regulated in pineal gland after HIBD and PC 12 cells after OGD.3.After miR-9a-up lentivirus transfected into PC 12 cells,the expression of miR-9a-5p was significantly higher than that of the control group(P<0.05);MT secretion was reduced,and the mRNA expression of clock,perl,cryl,and baml1 were lower than those of the control Cells(P<0.05).After miR-9a-5p inhibitor transfected into PC12 cells,the clock,perl,cryl,and baml1 mRNA expressions of miR-9a-5p inhibitor transfection+OGD group cells were higher than those of miR-9a-5p inhibitor control transfection+OGD Cells in group(P<0.05);MT secretion increased at 72h after successful transfection(P<0.05).4.After lv-rno-mir-9a-1 injection into the left lateral ventricle,the expression of miR-9a-5p was high and the target gene clock mRNA was low in pinecone detected by RT-qPCR in SD rats at the age of 3 months.5.In the Morris water maze experiment,the average escape latency time of rats gradually decreased after intensive swimming training.The number of crossing platforms was significantly reduced compared with the CON253 group(P<0.05).In the open-field experiment,the activity distance and frequency of entering the central region of rats in the mir-9a-up group was significantly greater than that in the CON253 group(P<0.05).There was no significant difference in the mean movement speed between the two groups(P<0.05).In the new object recognition experiment,the exploration time of the CON253 group was longer than that of the mir-9a-up group(P<0.05).In terms of the number of times to search for new objects,rats in the CON253 group were also more than those in the mir-9a-up group(P<0.05).The above shows that miR-9a-5p overexpression may damage brain tissue,emotional anxiety and reduce learning and memory abilities.Conclusion:1.After HIBD,the expression of miR-9a-5p was up-regulated in pineal gland,the expression of Aanat mRNA was down-regulated,and the secretion of MT was decreased.MiR-9a-5p may be involved in pineal gland damage after HIBD.2.The target gene of miR-9a-5p is clock,which is a component of mammalian biological clock.Combined with the abnormal secretion of MT,we found that after HIBD,miR-9a-5p affects the abnormal expression of other clock clock genes through targeted regulation of clock,thus participating in the pineal gland damage and functional abnormality.3.Overexpression of miR-9a-5p in the pineal gland of SD rats can lead to cognitive decline in learning and emotional anxiety,indicating that miR-9a-5p can participate in the pathophysics of pineal gland injury.