1. 10 th AOTA CONGRESS The study of microRNAs change in Iodine-induced autoimmune thyroiditis model of NOD.H-2 h4 mice ZHAO Shu-Jie, BI Mei, Fan Chen-Ling, LIU Miao, LIU Tong, LI Yu-Shu, SHAN Zhong-Yan, TENG Wei-Ping The Endocrine Department of the First Affiliated Hospital of China Medical University, the Endocrine Institute of China Medical University, the Liaoning Provincial Key Laboratory of Endocrine Diseases

2. 10 th AOTA CONGRESS Autoimmune thyroiditis (AIT) is one of typical organ specific autoimmune diseases. BACKGROUND Patients with AIT are characterized by lymphocytes infiltration in thyroid gland, destruction of thyroid follicular cells, serum autoantibodies such as TPOAb and TgAb in serum, and hypofunction.

3. 10 th AOTA CONGRESS In present opinion, immune cells (especially T cells and B cells) abnormality is immunological basis in AIT. Possible mechanisms contain: BACKGROUND Abnormal Th1/Th2 Functional deficiency of Ts 、 Treg Abnormal Th17 Cloned proliferation of autoreactive T cells and B cells

4. 10 th AOTA CONGRESS FITC-CD4 SSC-H APC-CD25 PE-Foxp3 Control 7.98% AIT 3.16% BACKGROUND Our previous study: CD4+CD25+Foxp3+ Treg cells in spleens from iodine-treated NOD.H-2 h4 mice and controls.

5. 10 th AOTA CONGRESS FITC-CD4 PE-IL-17 Control 0.89% AIT 2.45% BACKGROUND IL-17 expression in splenocytes from iodine-treated NOD.H-2 h4 mice and controls.

6. 10 th AOTA CONGRESS 结果 5 # # # # #:P<0.05 BACKGROUND Foxp3, IL-17 mRNA expression in splenocytes from iodine-treated NOD.H-2 h4 mice and controls.

7. 10 th AOTA CONGRESS MicroRNAs (miRNAs) have recently emerged as a major class of gene expression regulators linked to most biological functions. Several miRNAs have been identified as important regulators for immune cell development, as well as immune response. BACKGROUND

8. 10 th AOTA CONGRESS Recent studies have demonstrated that miR-155 plays a crucial role in the function of pathogenic immune cells, including Th1 cells, Treg cells, B cells, DCs and Th17 cells. BACKGROUND O’connell RM. Nat Reviews Immunol 2010,22: 111-122

9. 10 th AOTA CONGRESS It was reported that miR-326 is critical in regulating TH-17 differentiation and hence contributes to the pathogenesis of multiple sclerosis. Knockdown or overexpression of miR-326 alleviated or aggravated EAE, respectively. Martin AJ. Nat Immunol 2009,10: 1229-1231 BACKGROUND

10. 10 th AOTA CONGRESS BACKGROUND MiR-146a is among the miRNAs prevalently expressed in Treg cells and showed that it is critical for Treg functions. ZHOU L. Cell & Molecul Biological 2011,8: 380-381

11. 10 th AOTA CONGRESS OBJECTIVES The aim of this study was to observe the expression of miRNA-155, miRNA-146 and miRNA-326 in splenic CD4+ T cells and thyroid tissue of NOD.H-2 h4 mice with iodine-induced autoimmune thyroiditis.

12. 10 th AOTA CONGRESS MATERIAL AND METHOD 0.05% NaI solution （ 12week ） sterile water （ 12week ） 0.05% NaI solution （ 20week ） sterile water （ 20week ） iodine-treated groups （ n=10 ） control group （ n=6 ） iodine-treated groups （ n=10 ） control group （ n=8 ） get splenic CD4+T cells get thyroid tissue randomly divided NOD.H-2 h4 female mice at 4 weeks of age

13. 10 th AOTA CONGRESS Mice were sacrificed at the time point of 12 weeks after the beginning of experiment. CD4+T cells were be purified by MACS routinely separate mononuclear cells stained with multicolor immunofluorescence And detected by flowcytometry to analysis the percentage of CD4+CD3e+cells get spleen Target micoRNA Expression of splenic CD4+T cells were Measured by Real-time RT-PCR. The severity of lymphocytic infiltration was observed using HE-stained thyroid sections. get thyroid MATERIAL AND METHOD

14. 10 th AOTA CONGRESS Mice were sacrificed at the time point of 20 weeks after the beginning of experiment. get another thyoid Target micoRNA expression In thyroid were measured by Real-time RT-PCR. The severity of lymphocytic infiltration was observed using HE-stained thyroid sections. get thyroid homogenete MATERIAL AND METHOD

15. 10 th AOTA CONGRESS Table.1 The rate of thyroiditis of thyroiditis after NOD-2h4 mice were feed with sterile water and iodine water at the time point of 12 and 20 weeks （ % ） 12 weeks 20 weeks C ontrol 1000HI C ontrol 1000HI The rate of thyroiditis 0 (0/10) 100 # (6/6) 0 (0/10) 100 # (8/8) # P < 0.01 ， 1000 HI ： 0.05% NaI solution group(about 1000 times of normal daily iodine intake amount, 1000HI) Lymphocytic infiltration was observed in NOD.H-2 h4 mice fed with high iodine water for 12 weeks and 20 weeks (P < 0.01). RESULTS

16. 10 th AOTA CONGRESS Fig.1 HE-stained thyroid sections after NOD.H-2 h4 mice were fed with sterile water. (100X and 200X) RESULTS

17. 10 th AOTA CONGRESS Fig.2 HE-stained thyroid sections after NOD.H-2 h4 mice were fed with 0.05%NaI for 12week. (100X and 200X) RESULTS

18. 10 th AOTA CONGRESS Fig.3 HE-stained thyroid sections after NOD.H-2 h4 mice were fed with 0.05%NaI for 20 weeks. (100X and 200X) RESULTS

19. 10 th AOTA CONGRESS 0.41% FITC-CD4 PE- CD 3e 79.95% Fig.4 Splenic CD4+ Tcell separation ratio was analyzed by Immunofluorescence stain flow cytometric analysis. CD4+ Tcell (left) and rest of cells(right). RESULTS

20. 10 th AOTA CONGRESS groupnumbermean valueSDSE microRNA 155 iodine-treated 104.9744.3241.367 control 61.4180.96650.3946 microRNA 146 iodine-treated 103.3323.0850.9775 control 60.9650.93470.3816 microRNA 326 iodine-treated 102.5342.6660.8431 control 61.0390.39100.1596 tdfP Relative microRNA 1552.49910.4310.031 Relative microRNA 1462.26011.4810.044 Relative microRNA 3261.7429.6350.113 Table.2 12 weeks after the beginning of experiment. miRNA-155, miRNA-146, miRNA-326 in splenic CD4+ Tcells were quantified by realtime quantitative PCR. RESULTS

21. 10 th AOTA CONGRESS After 12th week, compared with control group （ 1.418±0.9665 ）, mice with AIT （ 4.974±4.324 ） have rised expression of miRNA-155 in splenic CD4+T cells (P < 0.05) RESULTS

22. 10 th AOTA CONGRESS After 12th week, compared with control group （ 0.9650±0.9347 ）, mice with AIT （ 3.332±3.085 ） have rised expression of miRNA-146 in splenic CD4+T cells (P < 0.05) RESULTS

23. 10 th AOTA CONGRESS After 12th week, compared with control group （ 1.039±0.3910 ）, mice with AIT （ 2.534±2.666 ）， there is no obvious change in expression of miRNA-326 in splenic CD4+T cells (P > 0.05) RESULTS

24. 10 th AOTA CONGRESS groupnumbermeanSDSE microRNA 155 iodine-treated 100.69290.28200.0892 control 80.17810.12340.0436 microRNA 146 iodine-treated 100.95290.26190.0828 control 80.26560.23500.0831 microRNA 326 iodine-treated 101.5850.40350.1276 control 80.51030.18980.0671 tdfP Relative microRNA 1554.786160.000 Relative microRNA 1465.85915.7370.000 Relative microRNA 3267.45613.3530.000 Table.3 20 weeks after the beginning of experiment. miRNA-155, miRNA-146, miRNA-326 in thyroid tissues were quantified by realtime quantitative PCR. RESULTS

25. 10 th AOTA CONGRESS After 20th week, compared with control group （ 0.1781±0.1234 ）, mice with AIT （ 0.6929±0.2821 ） have rised expression of miRNA-155 in thyroid tissues (P < 0.001) RESULTS

26. 10 th AOTA CONGRESS After 20th week, compared with control group （ 0.2656±0.2350 ）, mice with AIT （ 0.9529±0.2619 ） have rised expression of miRNA-146 in thyroid tissues (P < 0.001) RESULTS

27. 10 th AOTA CONGRESS After 20th week, compared with control group （ 0.5103±0.1898 ）, mice with AIT （ 1.585±0.4035 ） have rised expression of miRNA-326 in thyroid tissues (P < 0.001) RESULTS

28. 10 th AOTA CONGRESS Expression of miRNA-155, miRNA-146 and miRNA-326 were higher in both splenic CD4+ T cells and thyroid tissue of NOD.H-2 h4 mice with iodine-induced autoimmune thyroiditis than in control mice. miRNA-155, miRNA-146 and miRNA-326 may play important role in the pathogenesis of AIT. Further study are needed for over-expression or down-regulation of those microRNAs in AIT. CONCLUSIONS

29. 10 th AOTA CONGRESS