The Differentially Expressed MiRNAs In Nucleus Accumbens Of Methamphetamine Dependent SD Rats And The Target Genes Prediction And Verification

Objectives:Clarifying the mechanism of drug dependence is a key scientific issue in the scientific research of drug problem.This study intends to explore the differentially expressed microRNA(miRNA)and its target gene regulation mechanism in the nucleus accumbens of methamphetamine dependent SD(Sprague Dawley,SD)rats,so as to provide an indirect scientific basis for the methamphetamine dependent mechanism and provide a new target for subsequent withdrawal treatment.Methods:1 The methamphetamine dependent SD rat model was established as the administration group(ma4 group);rats in the control group were intraperitoneally injected with the same dose of normal saline(control group).The dependence of rats on methamphetamine was detected by conditional place preference(CPP)and stereotyped behavior experiment.The rats were killed to obtain the nucleus accumbens.The morphological changes of neurons and axons in the nucleus accumbens were detected by Glees silver staining and Hematoxylin Eosin staining.The number changes of neuron specific enolase(NSE)positive neurons and glial fibrillary acidic protein(GFAP)positive astrocytes in the nucleus accumbens of rats were detected by immunofluorescence assay.2 The miRNAs in rat nucleus accumbens were sequenced and analyzed,and the differentially expressed miRNAs were bioinformatics analyzed.The biological functions of the differentially expressed miRNA target genes were predicted by Gene Ontology(Go)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.3 The common target genes of miRNA with significant expression were screened by bioinformatics analysis software.Quantitative real-time polymerase chain reaction(QRT PCR)was used to detect the significantly expressed miRNA and its target gene messenger RNA(message RNA)in the nucleus accumbens of rats.Western Blotting(WB)was used to determine the expression level of the significant miRNA target proteins and the key proteins of Wnt signaling pathway.Results:1 Compared with the control group,the retention time of rats in MA4 group in the medicine box was significantly prolonged(P<0.05),and the stereotyped behavior score was significantly increased(P<0.05).In MA4 group,the neuronal axons in nucleus accumbens were distorted seriously,the polarization morphology of neurons disappeared and edema appeared.Glial cells proliferated obviously and formed glial nodules.The number of NSE positive neurons decreased significantly P<0.05),while the number of GFAP positive astrocytes increased significantly(P<0.05).2 MiRNA sequencing showed that 40 miRNAs were differentially expressed in the nucleus accumbens of MA4 rats,of which 17 miRNAs were up-regulated and 23 miRNAs were down-regulated.Go enrichment analysis showed that the differentially expressed miRNA target genes were mainly involved in cell component regulation,localization regulation,transport regulation and so on.KEGG enrichment analysis showed that the differentially expressed miRNA target genes were mainly focused on Wnt signaling pathway,spliceosome,lysosome and axon guidance.3 Compared with the control group,the expression levels of miR-217-5p,miR-31b,miR-28-3p,miR-31a-5p,miR-547-3p and miR-216b-5p in the nucleus accumbens of MA4 group were significantly up-regulated(P<0.05),while the expression levels of miR-1b,miR-144-5p,miR202-5p,miR-133a-3p,miR-133c and miR-451-5p were significantly down-regulated(P<0.05).Through bioinformatics analysis,five common target genes,namely 5-HTR1B,RBM8A,SYT7,RTN4 and SV2A,were screened from the above significantly expressed miRNAs.In MA4 group,the expression levels of 5-HTR1B,RTN4 and SV2A in the nucleus accumbens were significantly up-regulated(P<0.05),while the expression levels of Rbm8a and SYT7 were significantly down-regulated(P<0.05).The key proteins in Wnt signaling pathway were all significantly down-regulated(P<0.05),which were β-catenin,glycogen synthase kinase-3β,G1/s-specific Cyclin-D1,C-myc and Runt related transcription factor 2(Runx2).Conclusions:1 Methamphetamine changed the morphological structure of neurons and axons in the nucleus accumbens of SD rats.It is speculated that methamphetamine may cause neuroinflammatory reaction and oxidative damage by reducing the numbers of NSE positive neurons in the nucleus accumbens and promoting the proliferation of GFAP positive astrocytes.2 Enrichment analysis predicted that the differentially expressed miRNA target genes in nucleus accumbens were mainly involved in cell component regulation,localization regulation and transport regulation,focusing on Wnt signal pathway,spliceosome,lysosome and axon guidance.3 It is speculated that the differentially expressed miRNA in nucleus accumbens may inhibit the activity of Wnt signaling pathway and participate in methamphetamine dependent regulation by targeting the target genes and proteins of 5-5-HTR1B,RTN4,SV2A,Rbm8a and SYT7.