Research Of Microrna Expression Profile Of Peripheral Treg Cells And Uric Acid In AITD

BACKGROUNDWith the accelerated rhythm of life and work,the pressure is increasing.In recent years,the incidence of autoimmune thyroid disease(AITD) is also increasing year by year,Graves disease and hashimoto’s thyroiditis are common AITD,now most of the studies are focus on its pathogenesis. Many reports have claimed a role for CD4+CD25+T regulatory cells (Tregs) in the pathogenesis of Graves disease(GD), there was a deficiency of regulatory T cells in patients with active GD.MicroRNAs (miRNAs) are widely expressed in tregs,and are also involved in the regulation of immunity. We want to determine whether there were alterations of miRNA expression in peripheral treg cells, through the contrast between normal people and patients with Graves disease;And we also wished to provide clues for pathogenesis research of Graves disease,especially the mechanism of tregs’dysfunction,and reveal the role and significance of miRNA in autoimmune regulation.In addition,abnormal autoimmune regulation may cause series of abnormalities in cells,leading to metabolism disorders of glucose,lipid and nucleic acid etc.,and these disorders may further aggravate the abnormal autoimmune,forming a vicious cycle. For hashimoto’s thyroiditis of AITD,which exists hyperthyroidism for a short time,then hypothyroidism at the end, in all the causes lead to hyperthyroidism, hashimoto’s thyroiditis occupies considerable proportion. And other studies show that the abnormal thyroid function is correlated with the metabolism of purine nucleotide, both hyperthyroidism and hypothyroidism patients are found hyperuricemia,especially in hypothyroidism patients. All these indicate that AITD may piay an important role in the occurrence and development of abnormal purine nucleotide metabolism.For this reason, we further observed the clinical blood uric acid level in patients with hypothyroidism, analyzed the related factors,and discussed the relationship between the hypothyroidism and purine nucleotide metabolism.METHODSResearch one:There were15untreated patients with primary GD(group GD) and10normal people(group NC), record their histories, examination findings and general information in detail.All of them are volunteered to take part.The patients were randomly divided into3sub-groups (GD1,GD2,GD3),each group5people, female:male=4:1,10normal people were divided into3sub-groups (NC1、NC2、 NC3) too,one is4people,the other two are3, there’s1male in NCI1、NC2,there’s no male in NC3. Peripheral blood was obtained from each group,and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient separation,treg cells were isolated by immunomagnetic selection.The purity of the extracted cells was verified by the flow cytometry.RNA was extracted from tregs in each sub-group.In order to balance the individual differences,and reduce the error, RNA which extracted from the same sub-group was mixed as a sample pool, then for TaqMan low density miRNA Arrays analysis.The sub-groups in GD or NC group are considered as three biological replicates respectively,then we can use statistical method to analyse the microarray results.We have six sub-groups,six sample pools.accordingly six chips. Analyse miRNA array results of GD and NC group with statistical methods, find the differentially expressed miRNAs between the two groups, then to explore its relationship with autoimmune and GD.Research two:The levels of FT3,FT4,TSH, Anti-TG,Anti-TPO, TRAb, UA, TC, TG, LDL-C,HDL-C,Hcy, Cys, β2-MG,and24hUA were measured in36patients with hypothyroidism, compared with20healthy subjects,and analyzed the realated factors by univariate analysis.According to the exist of Anti-TG,Anti-TPO and TRAb, the patients were divided into two sub-groups:the autoimmune hypothyroidism(27cases) and the non-autoimmune hypothyroidism(9cases),then compared with each other.RESULTSResearch one:1.No statistically significant difference between the age groups;2.In GD group, FT3, FT4, TRAb were significantly higher than those of normal group,TSH was significantly lower than that of normal group, the differences were statistically significant.The differences of TGAb and TPOAb between GD and normal groups have not statistically significance.There’re no differences among GD1、GD2and GD3.There’re also no differences among NC1、NC2and NC3,there’s biological repeatability in each group.3. Flow cytometry show the purity of treg cells is88.96%;4.The results of TaqMan low density miRNA Arrays analysis:Compared with the normal group, there’re many miRNAs in GD group which are differentially expressed, among them significantly up-regulated with more than2times are:has-miR-106b、 has-miR-155and has-miR-181a, significantly down-regulated with more than2times are:has-miR-16and has-miR-146a;Analysis find these miRNAs are all associated with autoimmunity and GD。Research two:Compared with healthy subjects, patients with hypothyroidism had higher TSH,TC,TG,LDL-C,UA,Hcy,Cys,32-MG,and lower FT3,FT4,HDL-C,24hUA, the differences were significant (P<0.05);The univariate analysis showed that the UA of hypothyroidism has a significant positive correlation with TSH,TC,TG,Hcy,and is also negatively correlated with FT3,FT4,HDL-C;The thyroid function, autoimmune antibodies, TC, LDL-C, UA and24hUA also had significant differences between two sub-groups.CONCLUSION 1.This study is the first to show that there were statistically significant alterations of miRNA expression in peripheral tregs of GD patients.We speculate the differentially expressed microRNAs may influenced the immunity function of tregs, which leading to the occurrence of GD.2.We conducted a preliminary screening on relevant miRNAs in peripheral treg cells of GDpatients,with TaqMan low density miRNA array technology,this is the first time in domestic and overseas.The study may lay an important foundation for further study of occurrence and development mechanism in GD,it can also provides valuable clues. The results showed that has-miR-106b,has-miR-155,has-miR-181a,has-miR-16and has-miR-146a are differentially expressed in peripheral treg cells of GD.According to previous literature, has-miR-106b,has-miR-155and has-miR-146a have been confirmed realationship with treg’s regulatary function,and differentially expressed in a variety of autoimmune disease.While has-miR-181a and has-miR-16are new miRNAs which are discovered relationship with treg cells,this can provides an important way for the following research.3.Patients with hypothyroidism are more prone to hyperuricemia, The univariate analysis showed that the UA of hypothyroidism has a significant positive correlation with TSH,TC,TG,Hcy,and is also negatively correlated with FT3,FT4,HDL-C,all of these demonstrated that the hyperuricemia of hypothyroidism patients is related to decreased thyroid hormone, lipid metabolism disorder and hyperhomocysteine; The level of UA is higher in autoimmune hypothyroidism than in non-autoimmune hypothyroidism, which indicates that the autoimmune factor also play an important role in the occurrence of hyperuricemia.