Alterations And Validation Of MicroRNA Expression Profile Induced By Oxidative Stress In Mouse Hippocampal Neurons

Objective:With the development of technology and economy, the trendof aging increase rapidly in modern society. The morbidity of Alzheimer'sdisease (AD), the most prevalent neurodegenerative disorder, is rising yearafter year and influences on quality of life in early old age seriously. It ischaracterized by the reduction of neurons, intercellular neurofibrillary tangles,extracellular senile plaques composed mainly of Aβ peptides, and loss ofsynaptic function. As life expectancy increases, age-associated dementiasimpose heavy burdens on the individuals, families and society. However,efficient therapeutic strategies to treat AD are still missing, which is in partdue to the fact that the cause of AD is complex including gene mutation, aging,trauma and so on. The mechanisms underlying the pathogenesis of memoryimpairment in AD are oxidative stress theory, gene mutations theory,β-amyloid cascade theory, Tau protein hypothesis, mitochondrial dysfunctionand autophagy. Multiple lines of evidence suggest that progressive oxidativedamage is an early event involved in pathological process of AD. MicroRNAs(miRNAs) are a class of endogenous, approximately22nucleotide (nt),non-coding small RNA. It is well known that miRNAs play key roles inregulating target gene expression at the posttranscriptional level. A number ofstudies have witnessed that deregulated miRNAs play an important role indevelopment and prognosis of age-related neurodegenerative diseases. Butthere is no report about miRNAs alterations and the regulation mechanism oftarget gene in the early onset of AD induced by oxidative stress. Hydrogenperoxide (H₂O₂) is a stable, uncharged and freely diffusible reactive oxygenspecies. We stimulated the primary hippocampal neurons with H₂O₂todevelop a cell model of oxidative stress and studied the expression profiles ofoxidative stress-induced miRNAs. To clarify the roles of miRNAs in the process of oxidative stress leading to AD and its molecular mechanism, andprovided a basis for AD prevention and the development of miRNA targetingdrugs, the different expressed miRNAs were validated using real-time PCRassay and the bioinformatic analysis were performed in present study.Methods:(1) Animals: Healthy senescence accelerated mouse-resistant/1(SAMR1) of closed group and clean grade were purchased from LaboratoryAnimal Center of Peking University Health Science. The treatment of animalsin the experimental process was in accordance with the criteria of AnimalEthics.(2) Methods:â‘ Cell culture: Primary cultures of hippocampal neuronswere prepared from hippocampi of E18-E19SAMR1mice embryos. Theentire brain of fetal SAMR1mice was removed to a dish containing coldanatomy liquid and the hippocampi were separated completely. The meningesand vessels of hippocampi were isolated with microforceps. The hippocapalcells were plated onto poly-L-lysine-coated35mm dishes (5×108cells/L,2ml per dish),96-well culture (5×107cells/L,200μl per well) or24-wellcultuer (1×108cells/L,1ml per well) in fresh culture medium (DMEMsupplemented with20%FBS,100U/ml of penicillin and streptomycin),incubation at37℃in a humidified atmosphere of95%air and5%CO2. Aftercells attached to the substrate, the culture medium was changed to neurobasalmedium supplemented with2%B27,100U/ml of penicillin and streptomycin,followed by incubation for7days with half of the neurobasal medium beingchanged every3days to ensure cell maturation.â‘¡Identifications of primarycultured hippocampal neurons: At7DIV hippocampal cells were incubatedwith a monoclonal anti-MAP2antibody to identify neurons.â‘¢Cell viabilityassay: The primary cultured hippocampal neurons at7DIV were treated with0,100,200,400,800μM H₂O₂(Sigma) diluted with supplemented neurobasalmedium for24h at37℃in a humidified atmosphere of95%air and5%CO2.The cell viability was determined using the conventional3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.â‘£Measurement of cell apoptosis and death: According to MTT assay thesuitable H₂O₂concentration,200μM, was selected to treat the hippocampal neurons detecting the apoptotic cells by TUNEL staining and the dead cells byPI staining.⑤Microarray: RNAs from primary cultured hippocampal neuronsstimulated with0or200μM H₂O₂for6h were isolated with a MiRNeasyMini Kit (Qiagen). The miRNAs profiles from primary hippocampal neuronswere determined using GeneChip miRNA2.0Array (Affymetrix).â‘¥Bioinformatic analysis of miRNAs: According to the result of microarray,we selected miRNAs changed upon1.5times to perform bioinformaticanalysis using the DAVID (Database for Annotation, Visualization andInterrogated Discovery) bioinformatic resources.⑦Real-time PCR: Weperformed Real-time PCR to validate the result of microarray.⑧Statisticalanalysis: All data were presented as mean±SD. Statistical analysis wasdetermined by the independent Student t test and one-way ANOVA usingSPSS13.0software. A P value cut-off0.05was considered significant.Results:(1) At7DIV, neurons were matured in vitro and neuronaltubulin associated protein Map-2staining showed that the hippocampal cellshad developed dense netwoks indicative of fully differentiated and healthyneurons.(2) MTT assay revealed loss of cell viability significantly afterstimulating with H₂O₂for24h. Cell viability assay also showed thedose-dependent excitotoxicity of H₂O₂on primary cultured hippocampalneurons.(3) Primary cultured hippocampal neurons were subjected to TUNELassay. We found that the apoptosis rate of hippocampal neurons exposured to200μM H₂O₂for24h was increased significantly compared to that of normalcultured hippocampal neurons.(4) The result of PI staining showed that celldeath significantly increased with exposure to200μM H₂O₂for24h.(5) Theresult of GeneChip miRNA2.0Array showed that there were101deregulatedmiRNAs in H₂O₂-or control-treated primary hippocampal cells, in which17miRNAs changed above1.5time. The result of microarray showed that afterH₂O₂stimulation12miRNAs upregulated including mmu-mir-26b,mmu-mir-296, hsa-mir-9-1, mmu-mir-369, hsa-mir-200c, mmu-mir-32,mmu-mir-1965, hsa-mir-708, mmu-mir-1190, hsa-mir-377, mmu-mir-135b,mmu-mir-201;5miRNAs downregulated including mmu-mir-470, mmu-mir-713, mmu-mir-297c, mmu-mir-190b, mmu-mir-291a.(6) We usedDAVID platform to analyse functional annotation of deregulated miRNA-target genes. Bioinformatics and statistical analysis showed that thesemiRNAs may be involved in the regulation of cell growth, differentiation,apoptosis, neurotrophin transduction, signal transmission, cancer development,and so on.74target genes predicted by miR-708, miR-296, miR-200c,miR-377and miR-1190took part in activing the mitogen-activated proteinkinase (MAPK) signaling pathway.(7) Based on the bioinformatics analysisresults, pathology of AD and previous reports, we chose5miRNAs, miR-32,miR-196b, miR-26b, miR-708and miR-135b to perform real-time RT-PCRconfirmation. miR-135b and miR-708displayed a consistent upregulation inprimary hippocampal neurons with H₂O₂stimulation for6h.(8) Enrichmentfor Gene Ontology (GO) terms for biological processes and molecularfunctions of miR-135and miR-708targets were tested. Analysis of miR-135targrts revealed that miR-135related to DNA recombination, proteinubiquitination, protein amino acid autophosphorylation, transcription factorbinding, and so on. Result of miR-708targets showed that miR-708mailytook part in positive regulation of small GTPase mediated signal transduction,protein amino acid phosphorylation, positive regulation of cell proliferation,neuron development, etc.Conclusion:(1) The results of MTT assay suggested that the cellviability was significantly reduced after H₂O₂stimulation. The detection ofTUNEL and PI staining showed that the rate of cell apoptosis and deathobviously increased after H₂O₂stimulation. Our study demonstrated that theapoptosis of hippocampal neurons induced by oxidative stress may be one ofthe most important pathgenesis of AD.(2) Profiles of oxidative stress-relatedmiRNAs showed that the deregulated miRNAs may be represented inoxidative stress leading to AD in the process of the molecular mechanism.(3)The results of bioinformatic analysis provided more evidence that miRNAsinduced by oxidative stress may play important roles in the pathogenesis ofAD by influencing protein ubiquitination and phosphorylation through MAPK signalling pathway.