| Objective:Hashimoto’s thyroiditis（HT）,as a common organ specific autoimmune diseases,is the most important cause of hypothyroidism.Although the etiology of HT is not very clear now,with the increasing researches on HT in recent years,many studies have proved that Th17 cells are indispensable in the development of HT.The Th17 cell is a class of effector T cells characterized by secreting IL-17 cytokine family.Previous studies suggested that Th17 cells involved in the occurrence and development of autoimmune diseases,including multiple sclerosis（MS）,rheumatoid arthritis（RA）,systemic lupus erythematosus（SLE）,inflammatory bowel disease（IBD）.Several studies have demonstrated that IL-23/IL-23R/pSTAT3 signal pathway plays an irreplaceable role in the differentiation and maturation of Th17 cells.As a member of the IL-12 family,IL-23 plays a biological role by binding to the specific receptor on the cell membrane（m IL-23R）,to promote the phosphorylation of STAT3,and induce the differentiation and maturation of Th17 cells.It has been reported that a disintegrin and metalloprotease 17（ADAM17）can promote the ectodomain shedding of mIL-23R and inhibit the signal transduction of IL-23/IL-23R/pSTAT3.MicroRNAs（miRNAs）is a newly discovered endogenous single chain,noncoding small molecule RNA with a length of about 22nucleotides.It plays a major role in the post transcriptional regulation.It is involved in the regulation of gene expression to take part in a series of physiological and pathological processes.Mi R-326 is a member of numerous miRNAs.Previous studies have reported that miR-326 may take part in MS,experimental autoimmune encephalomyelitis（EAE）,SLE,type 1 diabetes mellitus（T1DM）and other autoimmune diseases.Our previous studies also found the elevated miR-326 level in NOD.H-2h4mice,a murine model of HT.And these results suggesting that miR-326 may be closely related with the occurrence of HT,but there is still lack of study in patients with HT.In the present study,we will analyze the level of miR-326 in thyroid tissue and peripheral blood of HT patients.We predicted that ADAM17 may be a target gene of miR-326 by bioinformatics analysis and ADAM17 may be involved in the nosogenesis of HT.The present study will provide a new idea and a therapeutic target for the diagnosis and treatment of HT in the future.Methods:In the present study,we recruited the patients undergoing thyroid surgery,who suffer from thyroid carcinoma or nodular goiter,and collected their thyroid tissue（the contralateral lobe of invalid thyroid or the tissue next to the thyroid nodule）.These tissue were divided into the Hashimoto group（HT group,n=24）and control group（non HT group,n=29）according to the pathological results and thyroid function.We also collected the peripheral blood of patients with HT（HT group,n=31）and healthy volunteers（NC group,n=23）.Their peripheral blood mononuclear cell（PBMC）,serum and plasma were isolated.Real time PCR（RT-PCR）were applied to detect the expression of mi R-326 and the related cytokines and transcription factors RNA expression of Th1/Th2/Th17/Treg cells in the PBMC and thyroid tissue.We also performed the flow cytometric analysis to detecte the percentage of Th1/Th17/Treg cells in peripheral blood.Enzyme-linked immunosorbent assay（ELISA）was used to detect the levels of IFN-γand IL-17A in serum or plasma.We also isolated the PBMC and cultured in vitro to polarize Th17 cells.Flow cytometric analysis the ratio of Th17 cells before and after polarization culture.In addition,to further explore the relationship between miR-326 and Th17 cells,we transfected miR-326 mimics to overexpress miR-326 in the cultured PBMC in vitro,and then detected the changes of Th17 cells.In order to further study the relationship between miR-326 and Th17 cells,we also collected peripheral blood of patients with HT（n=27）and healthy controls（n=32）,and separated PBMC and plasma.Flow cytometric analysis was used to explore the expression of ADAM17/mIL-23R in memory CD4+and memory CD8+cells.We also detected the expression of pSTAT3 in memory CD4+and memory CD8+cells before and after the stimulation with IL-23 in PBMC.We also detected the soluble IL-23R（sIL-23R）level in the plasma with ELISA.Specific inhibitor of ADAM17 were used in the cultured of CD3+T cells in vitro to explore the effects on the expression of mIL-23R.In addition,we also inhibited and overexpressed miR-326 in cultured PBMC separated from HT patients and healthy controls,to explore the regulation of miR-326 on ADAM17 and mIL-23R.Results:1.The expression of miR-326 and IL-17A were significantly increased in thyroid tissue and PBMC in patients with HT（P<0.05）,and miR-326 was positively correlated with IL-17 m RNA expression（P<0.05）.Flow cytometry analysis showed that the percentage of Th17 cells in the peripheral blood of HT patients increased significantly compared with that in normal controls（P<0.05）.2.After Th17 polarization culture in vitro,the proportion of Th17 cells increased significantly both in HT patients and NC group,and the degree of the increase in the HT group was significantly higher than that in the control group（P<0.05）.3.With the overexpression of miR-326 in vitro culture,the proportion of Th17 cells was significantly higher than that in the control group（P<0.05）.4.The mRNA and protein expression of ADAM17 in thyroid tissue and PBMC of patients with HT were significantly lower than the control group（P<0.05）,and the mRNA expression of ADAM17 was positively correlated with protein expression（P<0.05）.The RNA and protein expression of ADAM17 were both negatively correlated with miR-326 level（P<0.05）.Flow cytometry analysis showed that the expression of ADAM17 in CD4+and CD8+cell surface of HT patients（the mean fluorescence intensity,MFI）were significantly lower than that in the control group（P<0.05）.5.The expression of ADAM17 was significantly increased in CD4+T cells after inhibited miR-326 in the cultured PBMC separated from HT,and in contrast,the expression of ADAM17 was significantly decreased in PBMC separated from healthy controls after overexpressed miR-326（P<0.05）.6.The expression of mIL-23R on the surface of CD4+T cells was significantly higher than that in the control group（P<0.05）.The level of sIL-23R in HT patients in plasma was also significantly lower than that of the control group（P<0.05）.What’s more,the sIL-23R was positively correlated with MFI of ADAM17 in CD4+T cells（P<0.05）.7.After the inhibition of ADAM17 in CD3+T cells in vitro,the expression of mIL-23R was significantly elevated（P<0.05）.8.After IL-23 stimulation of PBMC,flow cytometry showed that pSTAT3 expression in CD4+T cells and CD8+T cells increased significantly compared with that before IL-23stimulation.What’s interesting is that the degree of increase in HT patients was much higher than that in control group.9.The expression of mIL-23R was significantly decreased,and the proportion of Th17 cells was significantly reduced（P<0.05）after inhibited mi R-326 in the cultured PBMC separated from HT,and the expression of mIL-23R and the proportion of Th17 were increased in the cultured PBMC separated from healthy controls after overexpressed miR-326（P<0.05）.Conclusion:1.Proportion of Th17 cells in HT patients increased significantly in the present study,which was consistent with previous studies.The percentage of Th17 cells in PBMC increased significantly after Th17 cell polarization culture.The degree of increase in HT group was significantly higher than that in NC group,suggesting that the ability of Th17 cell differentiation in HT patients is higher than that in control group.2.The expression of miR-326 in HT patients was higher than in healthy controls.The intervention culture in vitro suggested that miR-326 may has a positive regulatory effects on Th17 cells.3.The expression of ADAM17 in HT patients was significantly lower than that in the control group.After the overexpression or silence of miR-326 in PBMC,ADAM17 was significantly reduced or increased,suggesting that miR-326 had a negative regulatory effect on ADAM17.4.The expression of mIL-23R in HT patients was significantly higher than that in the control group.But the sIL-23R levels were significantly lower,and also positively correlated with ADAM17 levels.After ADAM17 inhibited by TAPI,the expression of mIL-23R increased significantly,which indicated the shedding of mIL-23R by ADAM17.5.After stimulation with IL-23,the rise degree of pSTAT3 expression in HT patients was significantly higher than that in the control group,which suggested that the high level of mIL-23R may be one of the reasons for the hyper-response of IL-23 in HT patients.6.The overexpression（or inhibition）of miR-326 experiments in cultured PBMC induced decreased（or increased）sIL-23R and elevated（or decreased）IL-17A levels.These results suggested that the positive regulation of miR-326 on Th17 cells can be achieved by targeting ADAM17 mRNA,reducing the ectodomain shedding of mIL-23R,indirectly increasing mIL-23R level in HT patients,and enhancing the signal transduction IL-23/IL-23R/pSTAT3 pathway. |
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