Research On The Relationship Between The Differentially Expressed MicroRMA In Hippocampus And Learning And Memory Of Rat After Microwave Exposure

Objective: Microwave is widely applied in many fields such as telecommunications,broadcasting and medical care. Companied with great benefits, it causes potentialharms to the health of the people. Studies have shown that microwave has asignificant effect on central nervous system while hippocampus is sensitive cephalicregion. The injury of learning and memory was important manifestations ofhippocampal damage induced by microwave exposure, but the mechanism of injury isnot yet clearly. MicroRNAs (miRNA) are a class of non-coding RNA endogenoussmall molecules involved in almost all major life activities, such as cell proliferation,apoptosis, growth, differentiation and metabolism. Studies have shown that thebrain-specific miRNAs were involved in the biological processes of mature neurons,including the development and differentiation of the nervous system and synapticplasticity. However, the study of the relationship between the differentially expressedmiRNA in hippocampus of rat after microwave exposure and learning and memorydysfunction has not been reported. Therefore, we detected the expression of miRNAsin rats’ hippocampus to screen the differentially expression of miRNAs aftermicrowave exposure, which is important to make clear the injury effects, and toclarify the mechanism and find out the sensation target.Materials and Methods：(1) In order to establish the animal models of learning andmemory damaged by microwave exposure, fifty-eight male Wistar rats were exposedto microwave with the average power density of30mW/cm2. The abilities of learningand memory were studied by the average escape latency in the Morris water maze.The contents of neurotransmitter were measured by HPLC. Morphological andultrastructural of rat hippocampus was observed by light microscopy and electron microscopy.(2) To screening the differentially expressed miRNAs in rats’hippocampus, the miRNAs expression profiles were detected by miRCURYTMLNAmiRNA microarray. It was maked that Fold change>1.5and P<0.05as standard toscreen differentially expressed miRNAs.(3) The targets of differentially expressedmiRNAs were predieted by the bioinformaties database (miRbase、miRanda andmiRDB). Then Gene ontology category and Kyoto Encyclopedia of Genes andGenomes (KEGG) pathway were used to analyze the function and pathways of targetgenes.(4) The relationship between miRNAs and learning and memory was studiedafter intensive analysis by Real-time PCR.Results:(1) The damaging effect of hippocampus by microwave exposure：Theaverage escape latency was longer significantly at6h and7d after exposure to30mW/cm2microwave compared with controls(P<0.05). The contents of Asp, Gluand GABA in hippocampus were decreased significantly at1d after exposure(P<0.01), and the contents of Asp and Gly in hippocampus were increasedsignificantly at7d after exposure (P<0.05). At1d after microwave exposure, thehippocampus tissue was edema. Few neurons show pyknosis and perivascular spaceget widened. The injuries were aggravated at7d after exposure, which shows thatmitochondrial swelling and cavitations, synapse less and blurring and postsynapticdensity decreasing. However, at14d it became recovery, and at28d it was basicallyrecovered.(2) The differentially expressed miRNA after microwave exposure:There were85miRNAs differentially expressed at7d after exposure,12miRNAsup-regulated and73miRNAs down-regulated, while18miRNAs differentiallyexpressed at14d after exposure,2miRNAs up-regulated and16miRNAsdown-regulated.(3) The result of target prediction and functional analysis: At7dafter radiation,44639,29918and7324target genes were predicted respectively bymiRbase, miRanda and miRDB, which had931common target genes. At14d,6780,6705and2273target genes respectively were predicted respectively by miRbase,miRanda and miRDB, which had155common target genes. The targets were involved in learning and memory, energy metabolism, proliferation and apoptosis,stress, cell cycle, signal transmission, blood brain barrier, ion channel and etc. Thesignaling pathways the target genes involved included neurotrophin signalingpathway, Circadian rhythm, Amphetamine addiction, Citrate cycle (TCA cycle),Synaptic vesicle cycle, long-term depression, tryptophan metabolism, Calciumsignaling pathway, serotonergic synapse, dopaminergic synapse and etc.(4) Theresult of Real-time PCR verification: rno-miR-125b-5p, rno-miR-338,rno-miR-125a-5p, rno-miR-9, rno-miR-181a and rno-let-7b were selected forintensive analysis by Real-time PCR. The results show that the candidated miRNAsare consistent with the microarray.(5) The functional analysis of the miRNAs:rno-miR-125b-5p, rno-miR-338, rno-miR-125a-5p, rno-miR-9, rno-miR-181a andrno-let-7b could affect the ability of learning and memory through ion channels,neurotransmitter secretion, apoptosis, mitochondrial activity and signal transductionthrough target genes.Conclusions:(1)30mW/cm2microwave exposure induced delayed average escapelatency, decreasing of neurotransmitter, neurons pyknosis and change of synapse inhippocampus. Those results suggested that we establish the animal models of learningand memory damage by microwave exposure successfully.(2) The expressions ofmiRNAs in hippocampus were changed sharply after microwave exposure, whichindicated that the differentially expressed miRNAs may be associated withhippocampus injury.(3) The targets of differentially expressed miRNAs wereinvolved in learning and memory, energy metabolism, proliferation and apoptosis,stress, cell cycle, signal transmission, blood brain barrier, ion channel and etc. Itsuggested that hippocampus injury induced by microwave was mediated by theregulation of target genes through miRNAs, and the regulation was multiple targets,multiple pathways and networked.(4) The results of Real-time PCR ofrno-miR-125b-5p, rno-miR-338, rno-miR-125a-5p, rno-miR-181a, rno-miR-9andrno-let-7b were consistent with those of the chip study. The target genes’ function and pathways of those miRNAs were closely related to learning and memory, whichindicated that those miRNAs is involved in regulating the effect of microwaveirradiation on learning and memory impairment.