The Role Of MiR-146a And Its Target MRNAs In The Pathogenesis Of Graves' Disease

Background: Graves' disease(GD)is a common autoimmune endocrine disorder except type I diabetes mellitus,as well as the most common cause of adult persistent hyperthyroidism.In some severe cases,patients would be dead for thyroid crisis and hyperthyroid heart disease.Due to little knowledge about the etiology and pathogenesis of GD,there is a lack of effective treatment for GD which has high recurrence rate.Therefore,further studies are required for exploring the etiology and pathogenesis of GD.MicroRNA-146a(miR-146a)is a new type of gene and immune regulatory factor in eukaryotes,which can participate in the pathogenesis of various autoimmune diseases by differential expression and directly reducing the expression of its potential target mRNAs or inhibiting their translation into proteins.Peripheral blood mononuclear cells(PBMCs)are key effector cells involved in the immune response.Determining the expression of miR-146 a and its immune-related target mRNAs in PBMCs of patients with GD is helpful to further elucidate the pathogenesis of GD.Objective: To investigate the expression and clinical significance of miR-146 a and its immune-related target mRNAs in PBMCs of patients with GD.Methods:1.Fasting EDTA-K2 anticoagulant venous blood samples from 30 patients with newly diagnosed GD,28 GD patients being treated,31 GD patients in remission period and 26 healthy donors were collected.PBMCs were isolated and purified from them by density gradient centrifugation and total RNAs in PBMCs were extracted by Trizol method.MiRNAs and mRNAs in total RNAs were retrotranscribed into corresponding cDNA by poly(A)-tailed and conventional reverse transcription methods,respectively.Quantitative real-time polymerase chain reaction(qRT-PCR)was performed to determine the relative expression levels of miR-146 a and its target mRNAs in PBMCs,and the correlation with them and clinical factors was also analyzed.Immune-related target mRNAs of miR-146 a include mRNAs of interleukin 8(IL-8),interleukin-1 receptor-associated kinase 1/2(IRAK1/2),tumor necrosis factor receptor associated factor 6(TRAF6),chemokine(C-C motif)ligand 5/8(CCL5 / CCL8)and Fas-associated protein with death domain(FADD).2.SPSS 24.0 was used for statistical analysis of the data.Mean±SD and Median(P25,P75)presented continuous measurement data conforming to normal and skewness distributin,respectively.Statistical methods include t-test, t'-test,Mann-Whitney U test,kruskal-wallis 1-way ANOVA test,Chi-square test and Spearman correlation analysis.P<0.05 was considered statistically significant in all tests.GraphPad Prism 5 was applied to draw the graph.Results:1.Compared with the healthy control group and the GD patient in remission group,the expression levels of miR-146 a in PBMCs in the newly diagnosed GD patients group and the patient group with GD being treated were both significantly decreased(P<0.05),but that of mRNAs of IL-8 and CCL8 were both significantly increased.(P <0.05).2.There was no statistically significant difference in the expression levels of miR-146 and mRNAs of IL-8 and CCL8 in PBMCs in between the healthy control group and the GD patient in remission group(P >0.05).3.Compared with the primary GD patient group,the expression level of mRNA of IL-8 in PBMCs in the patient group with GD being treated was decreased(P <0.05),while that of miR-146 a and mRNAs of CCL8 were not significantly different(P >0.05).4.Compared with the healthy control group,the relative expression levels of mRNAs of CCL5 and TRAF6 in PBMCs in primary GD patient group were significantly decreased(P<0.05),while that of IRAK1,IRAK2 and FADD were not statistically different(P>0.05).5.The expression level of miR-146 a in PBMCs was negatively correlated with that of mRNAs of IL-8 and CCL8(r=-0.294,r=-0.298,P<0.05),as well as serum total triiodothyronine(TT3),total tetraiodothyronine(TT4),free triiodo-thyronine(FT3),free tetraiodothyronine(FT4)and thyrotropin receptor antibody(TRAb)levels(r=-0.343,r=-0.347,r=-0.374,r=-0.409,r=-0.302,P<0.01),but positively correlated with serum thyroid stimulating hormone(TSH)(r=0.391,P<0.01).6.The expression level of mRNA of IL-8 in PBMCs was negatively correlated with serum TSH level(r=-0.549,P<0.01),but positively correlated with TT3,TT4,FT3,FT4 and TRAb(r=0.552,r=0.529,r=0.591,r=0.602,r=0.464,P<0.01).7.The expression level of mRNA of CCL8 in PBMCs was negatively correlated with serum TSH level(r=-0.325,P<0.01),positively correlated with TT3,FT3,FT4 and TRAb levels(r=0.215,r=0.217,r=0.215,r=0.370,P<0.05),but not significantly correlated with TT4 levels(P>0.05).Conclusion: Mir-146 a in PBMCs is closely related to GD,and it may be play a role in the pathogenesis of GD by inhibiting the expression of its target mRNAs of IL-8 and CCL8.Its expression level can be used as an auxiliary biological indicator to predict the disease state of GD,and may also become a biological target for the treatment of GD in the future.