Effect Of Autophagy Regulated By MiR-30a On Synaptic Plasticity Damage Induced By Microwave Exposure

ObjectiveMicrowave(300MHz-300GHz)played a more and more crucial role in many important fields of human society,and the health hazards that followed were of great concern.The brain was the most sensitive target organ to microwave exposure.Studies had shown that microwave exposure could induce synaptic plasticity damage in hippocampal neurons,but the mechanism was not yet clear.MicroRNA-30a(miR-30a)was a non-coding RNA differentially expressed in rats'hippocampus after microwave exposure,which was explored by our group.It had been reported that down-regulation of miR-30a could enhance autophagy mediated by Beclin1 and reduce ischemic brain damage.However,the relationship between miR-30a and autophagy in neurons and its role in synaptic plasticity after microwave exposure had not been studied up to now.Therefore,this study aimed to reveal the role of miR-30a in the regulation of autophagy in synaptic plasticity damage induced by microwave exposure,and to lay a foundation for elucidating the molecular mechanism,exploring the biomarker and targets of the prevention and treatment of brain injury induced by microwave exposure.Materials and methods1.Changes of miR-30a expression in rats'hippocampus synaptic plasticity damage induced by microwave exposure and its target genes110 male Wistar rats were randomly divided into sham exposed group and microwave exposed group,and exposed with 30mW/cm~2 microwave source for 15min every other day,which was repeated for 3 times in total.At 6h,7d,14d,1m and 2m after exposure,the expression of miR-30a in hippocampus was detected by Real-time PCR.The distribution of miR-30a in hippocampus was detected by in situ hybridization.The target genes of miR-30a were predicted by miRDB,miRWalk,miRanda and TargetScan,and the enrichment analysis of functions of the predicted target genes were performed with the KEGG.The interaction between miR-30a and its target gene AMPK(Prkaa2)was confirmed by dual luciferase reporter assays.Western Blot was used to detect the expression of AMPK(Prkaa2)expression,which was the target gene of miR-30a.2.Effects of microwave exposure on autophagy,miR-30a and its target gene AMPK expression in synaptic plasticity damage of PC12 cellsThe NGF-induced PC12 cells were exposed with 30mW/cm~2 microwave exposure for 15min.At 1h,6h,12h and 24h after exposure,the morphological structure of the cells was observed by scanning electron microscopy.The amino acid neurotransmitters were detected by HPLC.The morphological structure of autophagy was observed by transmission electron microscope.Western Blot was used to detect the expression of autophagy markers of Beclin1 and LC3-II/I(pretreated with 50?mol/L chloroquine).Real-time PCR was used to detect miR-30a expression and Western Blot was used to detect the AMPK(Prkaa2)expression,which was the target gene of miR-30a.3.Effect of miR-30a intervention on synaptic plasticity,autophagy and AMPK of PC12 cells after microwave exposureMiR-30a mimic was used to construct miR-30a overexpressing and its control PC12 cell model.Using 30mW/cm~2 microwave exposure,the morphological structure of PC12 cells was observed by scanning electron microscopy at 6h after exposure.Western Blot was used to detect the expression of autophagy markers Beclin1 and LC3-II/I(pretreated with 50?mol/L chloroquine)and AMPK(Prkaa2),the target gene of miR-30a.Results1.Changes of expression and distribution of miR-30a in rat hippocampusCompared with sham exposed group,the expression of miR-30a in hippocampus of microwave exposed rats decreased significantly at 7d,14d,and 1m after exposure(P<0.01).MiR-30a in hippocampus of rats in sham exposed group was positively expressed in the cytoplasm and nucleus of pyramidal cells in hippocampal DG region.On the 7d and 14d after exposure,miR-30a in hippocampus of microwave exposed rats was weak positively expressed in the cytoplasm and some nuclei of pyramidal cells in DG region,and their IOD and MOD were significantly reduced(P<0.01 or P<0.05).2.Prediction and validation of miR-30a target genesTarget genes of miR-30a were mainly involved in 11 signaling pathways(P<0.05):Glutamatergic synapse,Apelin signaling pathway,FoxO signaling pathway,Cellular senescence,cGMP-PKG signaling pathway,Oxytocin signaling pathway,Serotonergic synapse,AGE-RAGE signaling pathway in diabetic,Chagas disease(American trypanosomiasis),Autophagy-animal,Cytokine-cytokine receptor interaction.There were 8 target genes related to autophagy(P<0.05):Atg12,Ddit4,Pik3r2,Gabarapl2,AMPK(Prkaa2),Irs1,Beclin1 and Rras2.The activity of wild type AMPK(Prkaa2)luciferase was significantly decreased in the miR-30a mimic group(P<0.01),confirming that AMPK(Prkaa2)was the target gene of miR-30a.3.Changes of the AMPK(Prkaa2)in rats'hippocampusCompared with the sham exposed group,AMPK(Prkaa2)expression was significantly up-regulated in the microwave exposed hippocampus at 14d,1m,and 2m(P<0.01).4.The morphological structure of protrusion and changes of amino acid neurotransmitters of PC12 cellPC12 cells in the sham exposed group had elongated protrusion,and the cells were abundantly connected like a net.The PC12 cells in the microwave exposed group had a shorter protrusion at 1h after exposure,and the connection was reduced.Protrusion and connections were significantly reduced or even ruptured at 6h after exposure,and cell connections disappeared 24h after exposure.At 1h and 6h after exposure,the release of GABA increased significantly(P<0.01),which was an inhibitory amino acid transmitters.The release of Glu decreased significantly(P<0.01),which was an excitatory amino acid transmitters.The ratio of Glu/GABA decreased significantly(P<0.01).5.Changes of autophagic structures and Beclin1 and LC3-II/I expression in PC12 cellsOnly a few lysosomes were observed in the sham exposed group.A large number of autophagosomes and lysosomes were observed in the microwave exposed group at 6h after exposure,and there were still some autophagosomes and lysosomes observed in the microwave exposed group at 24h after exposure.Compared with sham exposed group,Beclin1 expression in microwave exposed group was significantly up-regulated at 6h after exposure(P<0.01).Without treatment with chloroquine,the ratio of LC3-II/I in microwave exposed group PC12 cells was not significantly changed 6h,12h,and 24h after exposure.However,with chloroquine pretreatment,LC3-II/I ratio was significantly increased at 6h and 12h after exposure(P<0.01).6.Changes of miR-30a and AMPK(Prkaa2)expression in PC12 cellsCompared with sham exposed group,the expression of miR-30a in microwave exposed group was significantly decreased at 6h after exposure(P<0.01),and AMPK(Prkaa2)was expressed significantly upregulated at 12h and 24h after exposure(P<0.01or P<0.05).7.Protrusion changes of PC12 cells after miR-30a overexpressionMiR-30a expression was significantly increased at 6h after microwave exposure in PC12 cells with mimic(P<0.01).In sham exposed and miR-30a negative control group had elongated protrusion,and the cells were abundantly connected like a net;in sham exposed and miR-30a overexpressing group,the protrusion were relatively short,and cells were in connection;while in microwave exposed and miR-30a negative control group,the protrusion were shortened or reduced;microwave exposed and miR-30a overexpressing group,the protrusion were shortened or reduced or even broken,and there was almost no connection between cells.8.Changes of autophagy markers in PC12 cells after miR-30a overexpressionBeclin1 expression in PC12 cells of miR-30a overexpressing group was significantly decreased at 6h after exposure(P<0.01).With chloroquine pretreatment,the ratio of LC3-II/I of miR-30a overexpressing group decreased significantly at 6h after exposure(P<0.01).9.Changes of AMPK(Prkaa2)in PC12 cells after miR-30a overexpressionAMPK(Prkaa2)expression in PC12 cells of miR-30a overexpressing group was significantly decreased at 6h after microwave exposure.(P<0.01).Conclusions1.30mW/cm~2 microwave exposed could induce the down-regulation and reduced-distribution of miR-30a in hippocampus of rats.2.MiR-30a target genes were mainly involved in 11 signaling pathways:Glutamatergic synapse,Apelin signaling pathway,FoxO signaling pathway,Cellular senescence,cGMP-PKG signaling pathway,Oxytocin signaling pathway,Serotonergic synapse,AGE-RAGE signaling pathway in diabetic,Chagas disease(American trypanosomiasis),Autophagy-animal,Cytokine-cytokine receptor interaction.There were 8 target genes related to autophagy,including Atg12,Ddit4,Pik3r2,Gabarapl2,AMPK(Prkaa2),Irs1,Beclin1 and Rras2.3.The dual luciferase reporter assay confirmed that AMPK(Prkaa2)was the target gene of miR-30a.30mW/cm~2 microwave exposure could upregulate the expression of AMK(Prkaa2)in hippocampus of rats.4.Microwave exposure could induce PC12 cells:(1)The synaptic structure and functional plasticity was damaged,which was manifested as shortened or reduced projections,unclear neurite structure,rough surface,abnormal amino acid neurotransmitter release.(2)Autophagy was up-regulated with an increase of autophagosomes,autophagy lysosomes,and increased expression of autophagy markers Beclin1 and LC3-II/I.(3)MiR-30a expression was decreased and its target gene AMPK(Prkaa2)expression was up-regulated.5.Overexpression of miR-30a resulted in aggravated synaptic plasticity damage in PC12 cells,shortened or even reduced of protrusions,decreased autophagy activity and autophagy markers expression,and decreased expression of AMRK(Prkaa2).MiR-30a could inhibit the expression of the target gene AMPK(Prkaa2),promote synaptic plasticity damage caused by microwave exposure,and inhibit the autophagy induced by microwave exposure.