Expression Profile Of MiRNAs In Peripheral Blood Of Patients With Hashimoto Thyroiditis And The Mechanism Of MiR-125a-5p

Objective:The second-generation high-throughput sequencing technology was used to analyze the expression profiles of peripheral blood mononuclear cells(PBMCs) miRNAs in patients with Hashimoto's thyroiditis(HT),using bioinformatics methods for predictive analysis and screening Identify miRNAs related to the pathogenesis of HT and further study its regulatory mechanism;explore the possibility of miRNA as a potential biomarker for HT and provide new ideas for the clinical diagnosis and treatment of HT.Methods:1.The RNA in peripheral blood mononuclear cells(PBMCs)of 5 HT patients and 5 healthy volunteers were detected by second-generation high-throughput sequencing technology,and the sequencing library was constructed.The expression profile of miRNAs was obtained by bioinformatics analysis.2.Six miRNAs that may be related to the pathogenesis of HT and have certain research value were selected from the expression profiles of miRNAs,the sample size was expanded,and the sequencing results were verified by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR).3.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)Pathways were used to analyze the functions of potential target Genes of differentially expressed miRNAs,and to predict their potential biological functions and biological processes.4.Target genes were predicted with Targetscan/miRanda database for miRNAs with significantly different expression in HT patients,and potential target genes involved in the pathogenesis of HT were screened out.5.The relationship between the expression level of miR-125a-5p and its target gene MAF mRNA was analyzed and verified in HT patients with PBMCs by qRT-PCR.In vitro,the dual luciferase reporter gene technology and interfering RNA technology were used to verify the targeting relationship between miR-125a-5p and MAF.6.Flow cytometry(FCM)and qRT-PCR were used to detect the differences in Th1 cell percentage and Th cell-related factor IFNG mRNA expression in PBMCs of peripheral blood between HT patients and healthy patients,and the relationship between miR-125a-5p expression level in PBMCs of HT patients and Th1 cell percentage and IFNG mRNA expression level was analyzed.7.Peripheral blood CD4~+T cells of healthy volunteers were isolated by magnetic bead separation and transfected with NC or miR-125a-5p inhibitor.The expression levels of miR-125a-5p,MAF mRNA and IFNG mRNA after transfection were detected by qRT-PCR.8.The relationship between the expression level of miR-125a-5p in PBMC of HT patients and the level of anti-thyroid peroxidase antibody(TPOAb)and anti-thyroid globulin antibody(TgAb)in serum was analyzed.qRT-PCR was used to verify the expression of miR-125a-5p,MAF mRNA and IFNG mRNA in thyroid tissues.9.ROC curve was used to analyze the diagnostic value of miRNAs with HT significantly differentially expressed.Results:1.The expression profile of miRNAs in PBMCs in peripheral blood of HT patients and healthy volunteers was analyzed by high-throughput sequencing technology.It was found that there were a total of 1310 miRNAs in the two groups,of which 651 were up-regulated and 659 were down-regulated.Among them,22miRNAs were significantly differentially expressed(Fold-change?1.5,p?0.05),12miRNAs were up-regulated and 10 miRNAs were down-regulated.2.Six miRNAs that may be related to the pathogenesis of HT and have certain research value were selected from the expression profiles of miRNAs,and their expression changes were verified by quantitative PCR.The results showed that the relative expressions of miR-132-5p,miR-301a-5p and miR-125a-5p were significantly up-regulated,while the relative expressions of miR-146b-3p were significantly down-regulated,which was consistent with the sequencing results.3.In patients with HT potential target genes differentially expressed micrornas in the biological function of prediction and analysis:GO analysis showed that among the up-regulated miRNA target genes,there were 24 GO terms that were significantly enriched in molecular function,13 GO terms that were significantly enriched in terms of cellular components,and 131 GO terms that were significantly enriched in biological processes;among the down-regulated miRNA target genes,there are 38GO terms that are significantly enriched in molecular function,30 GO terms that are significantly enriched in cell composition,and there were 199 GO entries that were significantly enriched in terms of biological processes.Kegg Pathways analysis showed that 18 signaling Pathways expressed up-regulated miRNAs target genes and16 expressed down-regulated miRNAs target genes might be involved.4.For high-throughput sequencing validation results consistent with the four miRNAs:miR-132-5p,miR-301a-5p,miR-125a-5p and miR-146b-3p,the prediction database analysis of potential target genes,combining with literature,screening potential target genes may be associated with the pathogenesis of HT MAF,and through the qRT-PCR verification,found that patients with HT PBMCs of MAF mRNA expression level significantly lowered(p<0.05),coinciding with increase of miR-125a-5p negatively correlated with expression level.Luciferase reporter gene confirmed the targeting relationship between miR-125a-5p and MAF gene.In vitro transfection experiments showed that compared with the transfected NC group,the expression of MAF mRNA and the cell proportion of MAF~+in healthy people with miR-125a-5p inhibitor group were significantly increased,suggesting that miR-125a-5p could inhibit the expression of MAF in a targeted manner.5.The percentage of Th1 cells in PBMCs of HT patients significantly increased compared with that of the healthy control group[(19.86±2.78)%vs(13.77±1.13)%,p<0.05].The relative expression level of IFNG mRNA of Th1 cells in PBMCs of HT patients was also significantly increased(0.22±0.21 vs 0.13±0.12,p<0.01).Correlation analysis revealed that the relative expression of miR-125a-5p in PBMCs of HT patients and the percentage of Th1 cells(r=0.4737;p=0.0224)was positively correlated with IFNG mRNA,but not with IFNG mRNA(r=-0.1429;p=0.5157).6.Relative expression levels of miR-125a-5p and IFNG mRNA in healthy CD4~+T cells transfected with miR-125a-5p inhibitor group were significantly lower than that of transfected NC,while relative expression levels of MAF mRNA were increased.In addition,the percentage of IFN-?~+T cells in CD4~+was significantly reduced,while the percentage of IL-4~+T cells was unchanged.7.By analyzing the relationship between miR-125a-5p and serum autoantibody level in peripheral blood of HT patients,it was found that the relative expression of miR-125a-5p was positively correlated with TPOAb level(r=0.4979,p=0.0156),but not with TgAb level(r=0.3926,p=0.0639).Compared with patients with simple goiter,the expression of miR-125a-5p in thyroid tissues of HT patients was significantly increased(0.17±0.13vs 0.05±0.03,p<0.05),and the expression level of IFNG mRNA in thyroid tissues of HT patients was also significantly increased(0.07±0.05vs 0.01±0.01,p<0.01),while the relative expression level of MAF mRNA in thyroid tissues of HT patients was significantly decreased(0.08±0.03 vs 0.19±0.13,p<0.05).8.ROC curve analysis evaluates the potential diagnostic value of miRNAs that are significantly differentially expressed in peripheral blood of HT patients.Compared with miR-301a-5p,miR-132-5p and miR-146b-3p,miR-125a-5p has AUC up to 0.744(95%CI=0.606-0.833,p=0.004)With a sensitivity of 72.91%and a specificity of 68.00%.Conclusions:1.The miRNAs expression profiles of PBMCs in peripheral blood of HT patients and healthy volunteers were screened and constructed by high-throughput sequencing.2.QRT-PCR was used to verify some differentially expressed miRNAs with research value,and the results of high-throughput sequencing were basically consistent,confirming the accuracy of the sequencing results.3.GO analysis and KEGG Pathyway analysis were used to predict the potential biological functions and possible biological processes of differentially expressed miRNAs in HT patients.4.MiR-125a-5p regulates Th1 cells by directly targeting MAF.In vitro interference with miR-125a-5p level in human peripheral CD4+T cells caused down-regulation of IFNG mRNA expression level and decreased proportion of CD4+IFN-+T cells.The expression level of miR-125a-5p in HT patients was significantly positively correlated with the level of patients'autoantibody TPOAb,which suggested that miR-125a-5p might be related to the occurrence of HT and reflect the severity of the disease to some extent.