Association And Regulatory Mechanism Of SNP And Methylation In NR3C1 And NR3C2 Genes With Schizophrenia

Objectives:The etiology of schizophrenia is complex and the pathogenesis is unknown.Due to the influence of the disease,patients are prone to suicide,self-mutilation and other self-injuring behaviors,as well as attacking others,destroying objects and other accident-causing behaviors that seriously threaten social and public safety,which bring huge mental pressure and economic burden to patient’s family and society.It is a major medical and social problem that has attracted much attention and needs to be solved urgently.At present,the diagnosis is mainly based on medical history and psychiatric examination,lack of objective biological indicators with reliable,efficient and reproducible.The diagnosis is susceptible to subjective influence,which increases the difficulty of forensic psychiatric identification.Genetic-environmental factors are the dominant factors in the pathogenesis of schizophrenia.Studies have shown that negative experiences in early life can lead to abnormal activation of the HPA axis,and HPA dysfunction is closely related to schizophrenia,depression and other psychiatric diseases,but the mechanism is not yet clear.Therefore,in this study,NR3C1 and NR3C2,two key receptor genes of the HPA axis,were used as target genes.The relationship between the SNP,promoter DNA methylation of NR3C1 and NR3C2 gene and schizophrenia,expression regulation mechanism and gene function were studied from the perspective of genetics and epigenetics.This study not only provides a theoretical basis for elucidating the pathogenic mechanism of schizophrenia,but also provides new ideas for the prediction,diagnosis and treatment and drug design of schizophrenia,and provides potential biological indicators for forensic psychiatric identification.Methods:1.The peripheral blood of schizophrenic patients and healthy controls were collected and genomic DNA was extracted.Five SNPs(rs6191,rs6198,rs6190,rs56149945,rs41423247)of NR3C1 and four SNPs(rs5522,rs5525,rs2070951,rs2871)of NR3C2 were selected for study according to the domestic and foreign literature reports,NCBI db SNP and HapMap database(MAF>0.1).The improve multiplex ligase detection reaction technology(iMLDR)was used for genotyping,and SPSS 22.0,SHEsis and MDR 3.0.2 software were used for statistical analysis.2.MicroRNAs(miRNAs)that may bind to the rs6191 in 3’-UTR region of NR3C1 gene were predicted by miRBase and SNP info database.The binding ability of rs6191 to miRNA was verified by dual-luciferase reporter gene system assay.The miR-422a mimics were added to cultured cells in vitro and the effect of miR-422a on the mRNA expression of NR3C1 gene was detected by real-time fluorescence quantitative PCR(RT-qPCR).RNA was extracted from peripheral blood of patients with schizophrenia and healthy controls.The mRNA expression level of NR3C1 gene and miR-422a were detected by RT-qPCR.The GR protein expression in serum was determined by Enzyme-linked immunosorbent assay(ELISA).3.The peripheral blood was collected from schizophrenia patients and healthy controls.Genomic DNA was extracted using the QIAamp DNA Blood Mini Kit and bisulfite treated with EZ DNAMethylation-Gold Kit,DNA methylation of the promoter regions of NR3C1 and NR3C2 gene was detected by Illumina HiSeq platform using MethylTarget technology.The association between DNA methylation of NR3C1 and NR3C2 gene and schizophrenia,and the gene environment interaction was analyzed.4.The recombinant plasmids of NR3C1 and NR3C2 gene were constructed using methylated vector.The plasmids were methylated by SssI methyltransferase in vitro,and the effect of methylation on promoter transcriptional activity was studied by dual-luciferase reporter gene system assay.The candidate functional site-binding transcription factors were predicted by JASPAR database.The effects of CpG sites on transcription factor binding were confirmed by Electrophoretic mobility shift assay(EMSA).Cells were treated with methylation inhibitor 5’-azabine to study the effect of demethylation on the mRNA expression levels of endogenous NR3C1 and NR3C2 genes.The mRNA expression level of NR3C2 gene in peripheral blood was detected by RT-qPCR,and the protein expression level in serum was detected by ELISA.5.SH-SY5Y cells were induced to differentiate by retinoic acid(RA).The length of neurite was measured by Image J software.And the mRNA expression level of MAPT,MAP2,SYP,NR3C1 and NR3C2 were detected by RT-qPCR.Then,RT-qPCR and WB were used to detect the knockdown efficiency of NR3C1 and NR3C2 genes in different cell lines.After knockdown,cell was induced with RA,the length of neurite was measured by Image J software.RT-qPCR was used to detect the mRNA expression level of marker genes.SH-SY5Y cell proliferation was detected by CCK-8 with knockdown of NR3C1 and NR3C2 gene.6.The differentially expressed genes after knockdown of NR3C1 and NR3C2 genes in SH-SY5Y cells were analyzed by RNA-seq.Ten genes were randomly selected from the top 30 differentially expressed genes,and the results of RNA-seq were verified by RT-qPCR.Go and KEGG analysis were used to explore the possible biological functions of NR3C1 and NR3C2 genes in schizophrenia.The possible biological functions of NR3C1 and NR3C2 genes were investigated by GO and KEGG analysis.Results:1.The rs6191 located in the 3’-UTR of NR3C1 gene is associated with female schizophrenia,and the risk of schizophrenia in female population carrying CC genotype is higher than AA or AC genotype.Frequencies of allele and genotype in rs2871,rs5522,rs5525 and rs2070951 of NR3C2 gene showed no statistical difference between the two groups.There was a strong linkage imbalance between rs5522 and rs5525(D ’=1.000,R2=0.974),and there was no significant difference in haplotype.There was interaction between SNP sites of NR3C1 and NR3C2 gene,rs6191/rs5522/rs2871 was the best model,and the consistency of cross validation was 10/10.2.Bioinformatics analysis predicted 7 miRNAs(hsa-miR-574-5P,hsa-miR-422a,hsa-miR-1183,hsa-miR-8485,hsa-miR-362-3p,hsa-miR-377,hsa-miR-570)may bind to the rs6191.Dual-luciferase reporter gene system assay showed that miR-422a inhibited firefly luciferase expression,and the inhibition degree in wild type was significantly higher than mutant type.When miR-422a was overexpressed in cells,the expression of NR3C1 was inhibited;when miR-422a inhibitor was added,the expression of NR3C1 was upregulated.This may suggested that miR-422a could regulate the expression of NR3C1.The mRNA and protein expressions of NR3C1 gene in schizophrenia patients were significantly lower than healthy controls.And the expression of miR-422a was significantly higher than healthy controls.From the SZDB database we found that NR3C1 gene expression level was decreased in the hippocampus,prefrontal cortex and striatum of schizophrenic patients.3.The methylation levels of NR3C1-1B and NR3C2-4 in women with schizophrenia were significantly higher than that in healthy controls.And significant differences in methylation at multiple CpG sites were found with gender specificity.The risk of schizophrenia was higher in the low education and unmarried groups than in the high education and married groups.Educational attainment,marital and smoking status among schizophrenics were correlated with methylation levels.4.The methylation of NR3C1-1H,NR3C2-2 and NR3C2-4 inhibited the transcriptional activity of genes.The JASPAR database predicted multiple transcription factors that might bind to candidate CpG sites.EMSA results showed that candidate CpG sites could affect the binding of transcription factors.The mRNA expressions of NR3C1 and NR3C2 were up-regulated after 5-azabine treatment.The mRNA and protein expression level of NR3C2 gene in schizophrenia patients were significantly lower than that in healthy controls.The expression of NR3C2 gene in the frontal cortex of schizophrenia patients was lower than healthy controls from SZDB database.5.According to the PsychENCODE database,we found NR3C1 and NR3C2 genes were up-regulated during fetal brain development.RA could induce SH-SY5Y cell differentiation,and the neurites were significantly prolonged.The mRNA expression levels of MAPT,MAP2 and SYP genes were significantly up-regulated,indicating that the SH-SY5Y neural differentiation cell model was successfully established.After knockdown of NR3C1 and nr3c2 genes,the neurites of SH-SY5Y cells were significantly prolonged.CCK-8 results showed that knockdown of NR3C1 and NR3C2 genes could promote the proliferation of SH-SY5Y cells.6.RNA-seq showed that knockdown of NR3C1 and NR3C2 caused 388 and 296 differential gene expression,of which 72 differentially expressed genes were overlapped.Ten differentially expressed genes were randomly selected and verified by RT-qPCR,which were consistent with RNA-seq results.GO and KEGG analysis showed that differentially expressed genes mainly involved in cell connection,biological regulation,response to stress,development and other processes,and enriched in chemokine signal pathway,MAPK,signal transduction related to environmental information processing,development,cell proliferation,growth and death,neurodegenerative diseases and other processes.Conclusions:1.rs6191 located in the 3’-UTR region of NR3C1 gene is associated with schizophrenia in female population,which may mediate the occurrence and development of schizophrenia by regulating NR3C1 gene expression through miR-422a.2.DNA methylation of NR3C1 and NR3C2 gene is associated with schizophrenia and sex-specific.Hypermethylation is more likely to develop schizophrenia.Education and marital status are risk factors for schizophrenia and interact with schizophrenia to influence gene methylation levels.3.Methylation of NR3C1-1H,NR3C2-2 and NR3C2-4 inhibited the transcriptional activity of genes.Demethylation can promote the mRNA expression of NR3C1 and NR3C2 genes.4.The mRNA and protein expressions of NR3C1 and NR3C2 genes in peripheral blood of schizophrenia patients were significantly lower than that in healthy controls.And the trend of NR3C1 and NR3C2 gene expression were consistent with that in brain tissue from SZDB database.5.NR3C1 and NR3C2 genes may play important roles in regulating neural differentiation,neurite growth and cell proliferation.RNA-seq results indicated that NR3C1 and NR3C2 genes may mediate the occurrence and development of schizophrenia by influencing signal transduction,development,cell growth and death related to environmental information processing,MAPK and other signaling pathways.