Research On The Effect Of MiR-219on Regulating NMDAR Signaling Pathway In The Synaptic Plasticity Injury Induced By Microwave Exposure

Objective: Microwave makes people’s lives easier and more convenient becauseof their applications in diverse fields. Meanwhile, there has been growing publicconcern regarding the adverse health effects associated with increased microwaveexposure. It could injure multiple functions of brain including the synaptic plasticityimpairment, but the underlying molecular mechanism remains largely unknown.MicroRNA (miRNA), non-coding RNA sequences, participates in diverse biologicprocesses, such as regulating the synaptic plasticity through affecting theN-methyl-D-aspartate receptor (NMDAR) signaling pathway. However, the roles ofmiRNA in microwave induced synaptic plasticity injury remain unclear. Therefore,this study could provide new evidence for understanding the molecular mechanism ofsynaptic plasticity impairment induced by microwave radiation, searching thebiomarkers and the targets of prevention and treatment.Materials and methods:(1)48male Wistar rats were exposed to microwave at0and30mW/cm2for10min every other day for three sessions. Rats were sacrificedat1,7,14and28d after the last microwave exposure. The structure of rats’hippocampus and synapse ultrastructure were observed by H&E staining andtransmission electron microscope. The expression of several key NMDAR signalingmolecules in hippocampal tissues was evaluated by Western Blot, including NR1,NR2A, NR2B, CaMKIIγ, CREB and p-CREB. NR1, NR2A, NR2B mRNAexpression and miR-219expression were detected by Real-time PCR. The expressionof miR-219in hippocampal tissues was also detected by in situ hybridization.(2)NGF-differentiated PC12cells were exposed to0and30mW/cm2microwave for5min and the synapse morphology was visualized with scanning electron microscopeand atomic force microscope6h after exposure. The release of amino acidneurotransmitters was determined by HPLC. Then the calcium influx inNGF-differentiated PC12cells was detected immediately after microwave radiationby using laser confocal microscopy and flow cytometry. The expression of NR1,NR2A, NR2B, CaMKIIγ, CREB and p-CREB was detected by Western Blot at1,6,12and24h after exposure. NR1, NR2A, NR2B mRNA expression and miR-219expression were detected by Real-time PCR.(3) Three miRNA target predictiondatabases, TargetScan, miRanda and miRDB, were used to infer the target genes ofmiR-219. The target genes analyses were carried out using gene ontology (GO)category and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.The target gene of miR-219was confirmed by Dual-Luciferase Reporter Assay System. Then the miR-219over-expression and silencing vectors were stablytransfected into PC12cells. The expression of miR-219target gene and NMDARsignaling molecules was detected by Western Blot at6h after microwave radiation.Results:(1) The changes of hippocampus structure and synapseultrastructure: At1d after microwave exposure, the hippocampal neurons weresignificantly injured; symptoms included degeneration, irregular arrangement,enlarged pericellular space and karyopyknosis with dark staining. The damages of thehippocampal neurons and synapses became more serious at7d after exposure. Thenumber of synaptic vesicle was decreased, and synaptic cleft became blurred. Thestructural injury was partially recovered14d after microwave exposure, and returnedto normal structure at28d after microwave radiation.(2) The changes of themiR-219distribution and expression in hippocampal tissues: In the sham group,the miR-219expression could be detected both in neuronal cytoplasm and nucleus ofhippocampus granular and pyramidal neurons.7d after exposure, the expression ofmiR-219was down-regulated (P<0.05or P<0.01), though there was no significantchange at14d after exposure.(3) The expression changes of NMDAR signalingmolecules in hippocampal tissues: The protein expression of NR1, NR2A, NR2Band CaMKIIγ was increased at1d after exposure (P<0.05), but the expression ofp-CREB was decreased7d after microwave radiation (P<0.05). The mRNAexpression of NR2A and NR2B was notably up-regulated (P<0.05or P<0.01).(4)The cell model of synaptic plasticity impairment: The synaptic length ofNGF-differentiated PC12cells shortened and the number of synapses decreased6hafter microwave exposure. The membrane surfaces of synapses became rough. Therelease of Gly, Glu and GABA was decreased (P<0.05), but the ratio of Glu andGABA was increased (P<0.05). And intracellular calcium level was increasedimmediately after microwave radiation in NGF-differentiated PC12cells (P<0.01).(5)The expression changes of NMDAR signaling molecules in PC12cells: Theexpression of NR1, NR2A, NR2B, CaMKIIγ and p-CREB was increased1h and/or6h after exposure (P<0.05). And the mRNA expression of NR1, NR2A and NR2B wasup-regulated (P<0.05or P<0.01).(6) miR-219expression in PC12cells: theexpression of miR-219was down-regulated at6h and12h after exposure (P<0.05orP<0.01).(7) Target gene prediction and analyses: The predicted fourteen targetgenes of miR-219, were involved in the synaptic plasticity related biologicalprocesses, including calcium ion transport, neurotransmitter transport, glutamatesecretion, long-term potentiation and long-term depression. Seven of those genes wereinvolved in the synaptic plasticity related pathways.(8) Confirming the target geneof miR-219: CaMKIIγ was a direct target of miR-219confirmed by usingDual-luciferase assay (P<0.05).(9) The regulation between miR-219and CaMKIIγand the expression of NMDAR signaling molecules in miR-219stable over-expressed and silenced cells: miR-219over-expression inhibited the expressionof CaMKIIγ, NR1, NR2A and p-CREB (P<0.05), while miR-219stable silencingincreased level of CaMKIIγ, NR1and p-CREB (P<0.05or P<0.01) in PC12cells at6h after exposure.Conclusions:(1)30mW/cm2microwave exposure induced the rat’shippocampus injury, particularly the synaptic plasticity impairment, and the activationof NMDAR pathway participated in the process.(2) Fourteen target genes of miR-219associated with synaptic plasticity were predicted, and seven of them were involved inthe synaptic plasticity related pathways. The down-regulation of miR-219induced by30mW/cm2microwave exposure could enhance the injury of synaptic plasticitythrough increasing the expression of miR-219target gene.(3)30mW/cm2microwaveexposure injured the synaptic plasticity of NGF-differentiated PC12cells. MiR-219downregulation could enhance the activation of NMDAR pathway via up-regulatingthe expression of CaMKIIγ, which might be a crucial mechanism about the synapticplasticity impairment induced by microwave.