The Effects Of MicroRNA132 On The Apoptosis In Oxygen-glucose Deprived Hippocampal Neurons

Objectives:Neonatal hypoxic-ischemic encephalopathy(HIE)is a disease of the central nervous system of newborns caused by a variety of perinatal factors including hypoxia in the head and decreased or even suspended cerebral blood flow.It is one of the main causes of neonatal acute death and long-term neurodevelopmental disorder.The pathogenesis of HIE is not yet clear,and the relevant clinical treatment methods are not ideal,and some children may have sequelae,or even lead to children’s disabilities.In the in vitro experimental study on the pathogenesis and treatment of HIE,the oxygen-glucose deprivation model of neurons can simulate the hypoxic-ischemia condition in vivo and has been widely used.MicroRNAs(miRNAs)are a class of endogenous non-coding single-stranded small RNAs with a length of 20～24 nucleotide(NT),which are widely expressed in the nervous system and are closely related to the development,differentiation and physiological function of nerve cells.After neuron injury due to hypoxia and ischemia,the expression of miRNAs can change and affect the expression of corresponding target genes,thus affecting cell apoptosis through various pathways.Therefore,miRNAs are the regulatory molecules of neuronal apoptosis and play an important role in neonatal HIE.At present,miRNAs have been intensively studied in the field of tumor diseases.There are also many experimental studies on the regulation of miRNAs on neurons in neurodegenerative diseases such as Alzheimer’s disease.However,there are limited studies on miRNAs in the field of HIE.In this study,microRNA132(miR-132),which is highly expressed in the nervous system,and oxygen-glucose deprived neurons in the nervous system were selected for experimental analysis to explore regulatory effect of miR-132 on neuronal apoptosis after hypoxic-ischemia in HIE.Methods:1.Culture the primary hippocampal neurons of fetal mice.Make the model of oxygen-glucose deprivation(OGD)of neurons.Detect the expression changes of miR-132 in this model by RT-qPCR.2.Transfect neurons by lentivirus transfection technique.Overexpress or down-regulated the expression level of miR-132 gene in neurons.Then the neurons are oxygen-glucose deprived.3.The interaction of miR-132 with FOXO3a and GRIA2 was detected by double luciferase reporter gene assay.The mRNA expression levels of FoxO3a and Gria2 after lentivirus transfection and oxygen and glucose deprivation were detected by RT-qPCR,and the protein expression levels of FoxO3a and Gria2 after lentivirus transfection and oxygen and glucose deprivation were detected by Western blot.Results:1.In some neurons,nuclear pyropysis,cell shrinkage,even nuclear fragmentation and dissolution,and cell body collapse.OGD model are successfully established.The expression of miR-132 was lower by qRT-PCR detected.With the extension of OGD time,the expression of miR-132 lower more obviously(P<0.01),and the results were statistically different.2.The transfected neurons have green fluorescence,and the transfection rate>85%.After transfection and oxygen-glucose deprivation,apoptosis was detected by TUNEL.Compared with the negative control group,miR-132 transfection group showed more serious apoptosis,P<0.01.Compared with the negative control group,the degree of apoptosis was reduced in the up-regulated miR-132 transfection group(P<0.05),and the results were statistically different.3.By Double Luciferase reporter Gene Assay,the wild-type binding ratio of miR-132 mimics to FOXO3a and mutant binding decreased,P<0.05,the combination ratio of wild type and mutant type of miR-132 mimics with Gria2 decreased significantly P<0.05,the results showed statistical difference;The expression of mRNA was detected by RT-qPCR.The expression of FoxO3a in the down-regulated miR-132 transfection group was higher than that in the negative control group(P<0.01),and the expression of FoxO3a in the up-regulated miR-132 transfection group was lower than that in the negative control group(P<0.05),showing statistical differences.The expression of GRIA2 in the down-regulated miR-132 transfection group was lower than that in the negative control group(P<0.05),the results showed statistical difference.The expression of GRIA2 in the up-regulated miR-132 transfection group was not significantly changed compared with that in the negative control group.The expression of protein was detected by western blot.The expression of FoxO3a in the down-regulated miR-132 transfection group was higher than that in the negative control group(P<0.01),and the results were statistically different.The expression of FoxO3a in the up-regulated miR-132 transfection group was not significantly different than that in the negative control group.The expression of GRIA2 in the down-regulated miR-132 transfection group was lower than that in the negative control group(P<0.05),the results showed statistical difference.The expression of GRIA2 in the up-regulated miR-132 transfection group was not significantly changed compared with that in the negative control group.Conclusions:1.The expression level of miR-132 in hippocampal neurons decreased after OGD,and the decrease was more obvious with the prolonging of OGD.2.Down-regulation of miR-132 expression can aggravate the apoptosis of OGD-deprived hippocampal neurons,while up-regulation of miR-132 expression can reduce the apoptosis of OGD-deprived hippocampal neurons.miR-132 can act as a protective factor in oxygen-glucose deficiency of neurons,and play an inhibitory role in neuronal apoptosis.3.Changes in the expression level of miR-132 can affect the expression of its target genes FoxO3 and Gria2.The pathway of miR-132 regulating the apoptosis of OGD-deprived neurons may be realized through the regulation of target genes FoxO3 and GluR2.