| Schizophrenia,which ranks first in the burden of mental diseases in our country,is a serious polygenic genetic disorder with abnormal thinking,emotion and behavior.Although comprehensive genome-wide association studies have revealed that a large number of common genetic variants were associated with schizophrenia susceptibility,the etiology of schizophrenia remained unclear.Therefore,further researches are needed to explore more undetected genetic variants.In the field of genetic research on schizophrenia,microRNA(miRNA)has attracted more and more attention.Many studies have revealed that miRNA not only plays an important role in the development of human brain,but also affects the occurrence of brain diseases.MiRNA targets and regulates the expression of multiple genes and has the potential to elucidate the complex pathological process of mental diseases.Objectives:To analyze the expression of miRNA between patients with schizophrenia and healthy controls and predict the regulatory target genes of differentially expressed miRNA in patients with schizophrenia.In order to provide evidence for etiological studies of schizophrenia,explore the association between schizophrenia and single nucleotide polymorphisms of target genes.Methods:Case-control study design was applied for the first part of this study.Case group consisted of 15 patients with first-episode schizophrenia and healthy controls were 1:1matched with cases by gender and age.RNA was extracted from all individuals’peripheral blood,and the expression of miRNAs in the peripheral blood of the case control groups was detected by transcriptome next-generation sequencing(NGS)method.Differential expression miRNAs were screened,and Quantitative Real-Time Polymerase Chain Reaction(RT-qPCR)was used to detect the expression of the most significantly expressed miRNA in NGS test.Finally,bioinformatics method was used to predict the target genes of the differential expressed miRNA.The second part of this study was also case-control study design,including 761 patients with schizophrenia and 775 healthy controls.DNA was extracted from peripheral blood,and the genotypes of the target genes were detected by flight mass spectrometry,so as to explore the association between single nucleotide polymorphisms in the non-coding and functional regions of these genes and schizophrenia.SPSS24.0,Haploview4.2,SNPStats online and Graphpad Prims 5.0 were used for statistical analysis and graphing.Results:1.A total of 30 subjects(15 cases and 15 controls),with an average age of25.6±5.42,were included in the transcriptome next-generation sequencing section of this study.The results of miRNA expression showed that there were 138 differentially expressed miRNAs between 15 patients with schizophrenia and 15 healthy controls,among which 87 were up-regulated and 51 were down-regulated.According to Fold change value,Padj value and read count value,six miRNAs were selected:has-miR-328-3p,has-miR-92b-3p,has-miR-1908-5p,has-miR-4467,has-miR-6786-3p and has-miR-30e.Based on literatures reviewing,further analysis of has-miR-1908-5p and has-miR-30e was performed by RT-qPCR.The results of RT-qPCR showed that the expression level of has-miR-1908-5p in peripheral blood of patients with schizophrenia was higher than that of healthy controls(P=0.085),however the expression level of has-miR-30e was not significantly different between schizophrenia patients and healthy controls.Has-miR-1908-5p was finally chosen as the study miRNA in the second stage of this study.2.Thirty target genes of has-miR-1908-5p were co-existed in three target genes predicting databases(TargetScan7.2,miRDB and DIANA microT-V5.0).The mRNA expression level of these 30 genes was checked in the transcriptome next-generation sequencing results,and the mRNA of six genes among the 30 co-existed target genes were significantly down-regulated in patients with schizophrenia,which corresponded to the up-regulation of miRNA-1908-5p.Based on literatures about function and studies of these six genes,furthermore,four genes(CACNA2D2,CMIP,DBN1 and ZNF385A)were finally selected to explore the association between gene polymorphisms and schizophrenia.3.SNPs in non-coding and functional region of the four genes mentioned above were selected from NCBI-SNP、Ensembl and 1000 Genome databases.Fifteen SNPs were selected to analyze the association with schizophrenia:CACNA2D2(rs35908181,rs45536634,rs59336240,rs12496815,rs3806706,CMIP(rs12925980,rs3751859,rs2287112,rs7193107,rs77700579),DBN1(rs2544809,rs2545794)and ZNF385A(rs2700159,rs2887875,rs2706252).4.The detection rates of rs35908181,rs59336240,rs45536634,rs3806706 and rs12496815 in CACNA2D2 gene were 97%,93%,97%,98%and 92%respectively.The results of Hardy-Weinberg Equilibrium(HWE)showed that the genotype frequency of rs59336240 and rs35908181 didn’t conform to HWE in control group(P<0.05),and other three SNPs conformed to HWE.The association analysis was conducted in these three SNPs.The results showed that between Schizophrenia patients group and healthy control group:(1)There were significant differences in allele frequency distribution of rs45536634(χ~2=4.002,P=0.045)and the risk of schizophrenia was 1.304 times(OR=1.304,95%CI=1.005-1.691)higher in people who carried A allele when compared with people carried G allele.According to Akaike information criterion(AIC),the codominant genetic model was the best model of this SNP(AIC=2060.3),under this genetic model,people who caring GG+AA genotype have a higher risk of schizophrenia compared with people who carried GA(OR=0.698,95%CI=0.523-0.931).(2)There were no significant differences in allele frequency distribution of rs3806706(χ~2=1.436,P=0.231).The overdominant genetic model was the best model of this SNP(AIC=2091.8),and under this model,the distribution of genotype were significantly.People carried GC genotype have a higher risk of schizophrenia(OR=1.255,95%CI=1.022-1.538)than people who carried GG+CC genotype.(3)There were no significant differences in allele frequency distribution of rs12496815(χ~2=0.354,P=0.552).Dominant and overdominant genetic models have the same AIC(1945.1)of this SNP,but under both these two models,no significant differences were observed.(4)The haplotype analysis were conducted in two or three adjacent SNPs of CACNA2D2,results showed that there was no haplotype associated with schizophrenia.5.The detection rates of rs2287112,rs7193107,rs77700579,rs12925980 and rs3751859 in CMIP gene were 96%,96%,98%,98%,and 98%respectively.All SNPs conformed to HWE(P>0.05)in control group.Association analysis conducted in these five SNPs showed that between case and control groups:(1)The distribution of allele of rs2287112 was not showed significant difference(χ~2=0.914,P=0.3339),but there were significant difference in the distribution of genotype(χ~2=5.754,P=0.016)under the recessive genetic model(AIC=2045.9)which is the best model according to AIC.Further analysis indicated that people with GG genotype was associated with the risk of schizophrenia(OR=2.419,95%CI=1.175-4.977).(2)There were no significant differences in allele frequency distribution of rs719310,rs77700579,rs12925980 and rs3751859,the genotype distributions also showed no significant differences between case and control groups considering the best genetic models.(3)The haplotype analysis of nine different haplotypes comprised by adjacent2,3,4,and 5 loci in CMIP gene were conducted and the results showed that there was a statistical significant difference of the haplotype of rs12925980-rs2287711-rs3751859-rs77700579 between case and controls(χ~2=23.683,P=0.003),and the TTGT haplotype would decrease with risk of schizophrenia(OR=0.42,95%CI=0.19-0.94)..6.The detection rates of rs2544809 and rs2545794 in DBN1 gene were both 97%and they all conformed to HWE(P>0.05).Association analysis showed that the allele frequency distributions of rs2544809 and rs2545794 were not observed to be significantly different between the case and control group.There were no statistically significant differences in genotype frequency distributions between the case and control groups considering the best genetic models of these two SNPs.However,haplotype analysis of these two SNPs showed a significant difference between case and controls(χ~2=6.861,P=0.032)and people who carried CC haplotype have a higher risk of schizophrenia(OR=1.57,95%CI=1.07-2.30).7.The detection rates of rs2700159,rs2887875 and rs2545794 in ZNF385A gene were 93%,97%,and 94%respectively,and they all conformed to HWE(P>0.05)in control group.Association analysis showed that the allele frequency distributions of rs2700159,rs2887875 and rs2706252 were not significantly different between case and control groups and there were also no statistically significant differences in genotype frequency distribution considering the best genetic models.The haplotype analysis showed no significant difference in the three haplotypes consisted of the three SNPs between case and controls.8.The results of gene-gene interaction analysis showed that the best model was rs12496815 and rs3806706 which were located in CACNA2D2 gene,however,there was no association with schizophrenia under this model(P>0.05).Conclusions:(1)The expression level of miR-1908-5p in the peripheral blood of patients with schizophrenia was significantly higher than that of healthy controls.(2)Thirty target genes of miR-1908-5p were co-existed in TargetScan,miRDB and DIANA microT databases,and the mRNA expression level of four genes(CACNA2D2,CMIP,DBN1,ZNF385A4)were down-regulated which correspond to the up-regulation of miR-1908-5p.(3)The SNP rs45536634 and rs3806706 in CACNA2D2 were both associated with schizophrenia,people who carried A allele of rs45536634 or GC genotype of rs3806706 may increase the risk of schizophrenia.(4)rs2287112 in CMIP gene is associated with schizophrenia and GG genotype would increase the risk of schizophrenia.(5)The haplotype consisted of rs12925980-rs2287711-rs3751859-rs77700579 in CMIP gene is associated with schizophrenia,and the TTGT haplotype would decrease the risk of schizophrenia.(6)The haplotype consisted of rs2544809and rs2545794 in DBN1 is associated with schizophrenia and CC haplotype would increase the risk of schizophrenia. |
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