The Role Of MiR-124 And Its Target Genes In The Major Depressive Disorder And Antidepressant Response

BackgroundMajor depressive disorder(MDD)is a kind of mental illness with high risk,high recurrence,high disability,and high suicide,and seriously influences the quality of patients' life.miRNAs,the small non-coding RNAs about 20-22 nucleotides,can regulate gene expression at the post-transcriptional level.miR-124 is specifically expressed in the central nervous system and is one of the most abundant miRNAs in the adult brain.Based on our previous studies and other evidence,miR-124 was abnormally expressed in depression animal models,in postmortem brain and peripheral tissue of MDD patients,suggesting that miR-124 is probably to be an important biomarker and therapeutic target for depression.Therefore,on the basis of the above evidence,this study intended to further elucidate the biological functions of miR-124 in MDD and antidepressants by focusing on its molecular regulation mechanisms(eg.single nucleotide polymorphisms(SNPs)and methylation)and downstream target genes.Methods1.Three SNPs,including a functional SNP in MIR124-1(rs531564),a micro RNA-binding site SNP(rs10759)and a promoter SNP(rs951436)in RGS4,were genotyped in 225 MDD patients and 436 controls.Among MDD patients,147 received antidepressant treatment for 8 weeks.The 17-item Hamilton Rating Scale for Depression(HAMD-17)was used to regularly evaluate at baseline,week 2,week 4,week 6,and week 8.Multifactor dimensionality reduction(MDR)was performed to identify gene-gene interactions.2.Methylation study: Thirty-three MDD patients and 33 age-and gender-matched healthy controls were recruited.Methyl Target was used to detect the Cp G islands located in the promoters of the three miR-124 precursor genes(MIR124-1,MIR124-2,MIR124-3)from 2 k upstream to 1 k downstream.Lasso regression was used to screen the differential Cp G sites which were most relevant to MDD.Methylation score was calculated to construct the discrimination model.3.The weighted gene co-expression network analysis(WGCNA)was used to identify the most significant gene module and hub genes related to MDD in peripheral blood mononuclear cells(PBMCs)(n = 45).Thirty-three MDD patients and 41 healthy controls were recruited to validate the expression levels of hub genes using quantitative real-time polymerase chain reaction(qRTPCR).4.The genes in the hub module screened by WGCNA were intersected with miR-124 prediction target genes.After a series of screening,the regulatory relationships between mir-124 and its prediction target genes(MPC1,NEK9,PLEKHF1,SLC12A2,TSPAN15)were detected by luciferase reporter gene assays.qRT-PCR was used to detect the m RNA expression levels of the five target genes.Results1.There was no significant association with MDD in single-SNP analyses.MDR analysis revealed the gene-gene interaction between MIR124-1 and RGS4,which increased the risk of MDD(OR=1.67,95%ci =1.20-2.33,p=0.0024).There was a significant association in genotypic frequencies of rs951436 between the remitter and non-remitter patients(p=0.026,correction p=0.078).After 8 weeks of treatment,the rs951436 heterozygote carriers had threefold probabilities of achieving clinical remission as compared with homozygotes(odds ratio =3.00,95% confidence interval =1.33-6.76,p=0.007,correction p=0.021).2.Eleven Cp G islands and 222 Cp G sites were sequenced.Nine Cp G islands were hypomethylated in MDD patients,and 137 Cp G sites showed significant differences between MDD patients and the control groups.Seven candidate Cp G sites which were most associated with MDD were screened by LASSO regression,and their methylation levels were significantly down-regulated in MDD patients.The AUC value reached 0.905 using the methylation score to discriminate between MDD and controls.3.The blue module was the hub module which was most related to MDD(R2 = 0.95).The genes in this module were mainly enriched in oxidative phosphorylation pathways and purine ribonucleotide metabolism and biosynthesis,which wre related to MDD.qRT-PCR analysis showed that the ATP5G1 gene was significantly down-regulated in MDD patients(t =-2.89,p= 0.005).4.Luciferase reporter gene assays showed that MPC1,TSPAN15,NEK9,SLC12A2 and PLEKHF1 could all be regulated by miR-124.q PCR detected that there were no statistically significant differences in m RNA expression levels of these 5 target genes between MDD patients and healthy controls.Conclusions1.miR-124 might be involved in the pathogenesis of MDD by regulating its target gene RGS4.2.The methylation level of miR-124 was probably to be involved in the pathogenesis of MDD.Methylation levels in the promoter regions of the three precursor genes were significantly decreased in MDD patients,which might be related to the up-regulation of miR-124 expression.