Differential MicroRNA Expression in Peripheral Blood Mononuclear

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Differential MicroRNA Expression in Peripheral Blood Mononuclear Cells from Graves’ Disease Patients Rongjiao Liu,* Xinran Ma,* Lingyan Xu,* Dao Wang, Xiaohua Jiang, Wei Zhu, Bin Cui, Guang Ning, Dongping Lin, and Shu Wang Shanghai Clinical Center for Endocrine and Metabolic Diseases (R.L., D.W., X.J., W.Z., B.C., G.N., S.W.), Department of Endocrinology and Metabolism, Rui-Jin Hospital, Affiliated to Shanghai Jiao-Tong University School of Medicine (SJTUSM), and Laboratory of Endocrinology and Metabolism (R.L., X.M., L.X., B.C., G.N., S.W.), Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and SJTUSM, Shanghai 200025, China; Genetics of Development and Disease Branch (X.M., L.X.), National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892; and Department of Endocrinology and Metabolism (D.L.), Shanghai Ninth People’s Hospital affiliated Shanghai Jiao-Tong University School of Medicine, SJTUSM, Shanghai 200011, China

Context: Graves’ disease (GD) is a common autoimmune disease that affects the thyroid gland. As a new class of modulators of gene expression, microRNA (miRNA) have been reported to play a vital role in immune functions and in the development of autoimmunity and autoimmune disease. Objective: This study sought to characterize the different miRNA expression in peripheral blood mononuclear cells (PBMC) from GD patients and healthy individuals and examine their direct responses to T3 treatment. Methods: Forty-one patients who met criteria for initial GD, 13 GD patients in remission, and 35 healthy controls were recruited. Microarray was used to analyze the expression patterns of miRNA in PBMC obtained from initial GD patients and healthy controls. Three top-ranked miRNA were selected and validated by TaqMan-based real-time PCR in healthy controls, initial GD patients, and GD patients in remission. Furthermore, we cultured PBMC from healthy donors with or without T3 treatment to examine direct effects of T3 on selective miRNA. Results: There were sixteen miRNA expressed differently in PBMC from initial GD patients compared with normal subjects. Further analysis consistently showed that the expression of miR-154*, miR-376b, and miR-431* were suppressed in PBMC from initial GD patients. In addition, their expression levels were recovered in GD patients in remission. Meanwhile, T3 treatment could directly inhibit the expression of these miRNA in cultured PBMC from healthy subjects. Conclusions: The present work revealed that differentially expressed miRNA were associated with GD and T3 exposure, which might serve as novel biomarkers of GD and potential targets for GD treatment. (J Clin Endocrinol Metab 97: E968 –E972, 2012)

raves’ disease (GD) is a common antibody-mediated autoimmune disorder of the thyroid gland characterized by hyperthyroidism and diffuse goiter with or without the associated ophthalmopathy and dermopathy (1, 2). Peripheral blood mononuclear cells (PBMC) consist

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mainly of monocytes, T cells, and B cells and smaller amounts of NK cells and dendritic cells of both myeloid and plasmacytoid origin. During GD pathogenesis, these immune cells are able to infiltrate into the thyroid gland and produce autoantibodies against thyrocytes. The thy-

ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2012 by The Endocrine Society doi: 10.1210/jc.2011-2982 Received October 30, 2011. Accepted March 5, 2012. First Published Online March 28, 2012

* R.L., X.M., and L.X. contributed equally to this work. Abbreviations: FT3, Free T3; GD, Graves’ disease; miRNA, microRNA; PBMC, peripheral blood mononuclear cells; qRT-PCR, quantitative real-time PCR; sTSH, sensitive TSH; TGAb, thyroglobulin antibody; TPOAb, thyroperoxidase antibody; TRAb, TSH receptor antibody.

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roid gland is then abnormally activated in response to the autoantibodies, producing excessive thyroid hormone and causing hyperthyroidism. Hyperthyroidism has a profound physiological impact on multiple aspects, including stimulating thyroid growth and stimulating the immune responses in general, thus exacerbating GD pathogenesis and development (3, 4). The etiology of GD appears to be multifactorial, with genetic and environmental factors being involved in disease development (1). Thus, we sought for a systematic discovery of molecules involved in GD as novel biomarkers and potential treatment targets. As a new class of modulators of gene expression, microRNA (miRNA) attracted our attention. miRNA act as negative regulators of the protein-coding gene expression involved in multiple biological processes (5). miRNA have been reported to play vital roles in autoimmune diseases. Hezova et al. (6) found significantly increased expression of miRNA-510 and decreased expression of miRNA-342 and miRNA-191 in T regulatory cells of type 1 diabetic patients. In multiple sclerosis patients, miR-18b and miR-599 were found to be relevant in relapse whereas miR-96 might be involved in remission (7). Meanwhile, miR-21, miR-148a, miR-3715P, miR-1224-3P, and miR-423-5P were reported to be associated with systemic lupus erythematosus (8, 9). To our interest, differentially expressed miRNA have been reported between thyroid gland tumors and normal tissues (10). GD is an important thyroid-related autoimmune disease, but the significance of miRNA in GD has never been elucidated. In the present study, we used miRNA microarray, quantitative RT-PCR, and cell culture approaches to discover different miRNA expression patterns in PBMC from healthy subjects, GD patients, and GD patients in remission with the aim of identifying novel miRNA as biomarkers of GD and providing potential targets for GD treatment.


Subjects and Methods Subject criteria and experimental procedures Forty-one initial GD patients and 13 GD patients in remission were recruited from Ruijin Hospital and the Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine. All initial GD patients, without previous treatment, were newly diagnosed through standard clinical and laboratory examination. The clinical evaluation included patients’ history, physical examination, and radioactive iodine uptake test. The laboratory examinations included free T3 (FT3), FT4, and sensitive TSH (sTSH) as well as TSH receptor antibody (TRAb), thyroglobulin antibody (TGAb), and thyroperoxidase antibody (TPOAb). GD patients in remission had been treated with methimazole for at least half a year and maintained a euthyroid state with largely improved parameters compared with initial GD patients. Thirtyfive age-matched healthy individuals as a control group were also enrolled. Informed consent was obtained from all participants before the samples were collected. The ethical committee of Shanghai Jiao Tong University School of Medicine approved the study protocol. PBMC were isolated from all subjects, and RNA samples were extracted for microarray and quantitative real-time PCR (qRTPCR) analysis. The detailed methods are described in Supplemental Materials and Methods (published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org).

Cell culture and T3 treatment Fresh PBMC obtained from six healthy subjects were isolated by Isopaque-Ficoll gradient centrifugation and washed with RPMI 1640 twice. Then 5 ⫻ 106 cells were cultured on six-well culture plates in a culture medium (RPMI 1640 plus 10% FBS plus 100 U/ml penicillin plus 100 ␮g/ml streptomycin) with or without T3 (Sigma-Aldrich, St. Louis, MO; 100 nM) for 24 h or 7 d. After 24 h and 7 d, the cells were harvested, and RNA were extracted for qRT-PCR analysis.

Statistical analyses All P values are two-sided, and P ⬍ 0.05 is considered a statistically significant difference. All statistical calculations were performed by the SPSS version 13.0 between two groups. All graphics were performed by GraphPad software. Data are presented as mean ⫾ SD.

TABLE 1. Clinical characteristics of healthy controls, initial GD patients, and GD patients in remission n (male/female) Age (yr) FT3 (pmol/liter) FT4 (pmol/liter) sTSH (mIU/liter) TRAb (IU/liter) TPOAb (IU/ml) TGAb (IU/ml)

Healthy controls 35 (10/25) 32.11 ⫾ 6.90 4.09 ⫾ 0.86 13.19 ⫾ 1.58 1.51 ⫾ 0.63 0.49 ⫾ 0.27 4.29 ⫾ 1.38 13.42 ⫾ 3.44

a

P ⬍ 0.01, initial GD compared with healthy controls.

b

P ⬍ 0.01, GD in remission compared with initial GD.

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Initial GD 41 (14/27) 33.75 ⫾ 12.65 29.88 ⫾ 15.33a 39.36 ⫾ 12.84a 0.001 ⫾ 0.001a 10.45 ⫾ 8.77a 253.81 ⫾ 223.70a 198.65 ⫾ 113.14a

GD in remission 13 (4/9) 35.36 ⫾ 10.90 4.71 ⫾ 0.76b 13.14 ⫾ 2.75b 1.92 ⫾ 0.82b 3.55 ⫾ 2.55b 29.1 ⫾ 28.4b 8.86 ⫾ 8.83b

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Results Clinical characters of GD patients and normal subjects Subject characteristics and anthropometric parameters are summarized in Table 1. A total of 89 participants including 41 initial GD patients, 13 GD patients in remission, and 35 healthy individuals were recruited in this study. The initial GD patients group had significantly higher serum FT3, FT4, TPOAb, TRAb, and TGAb levels and significantly lower serum sTSH level than normal subjects. The GD patients in remission maintained euthyroid states with largely improved parameters compared with initial GD patients.


on the fold changes and P value (fold change ⫽ ⫺6.47, ⫺5.57, and ⫺4.85; P value ⫽ 0.017, 0.001, and 0.008, respectively) between two groups. RNU48 was applied as internal control. Consistent with the microarray data, we found that the expression levels of miR-154*, miR-376b, and miR-431* in PBMC were reduced in GD and significantly recovered in GD remission (Fig. 1B). Moreover, these miRNA were correlated significantly with FT3, FT4, and TRAb levels (Supplemental Table 1).

Differential miRNA expression in PBMC from GD patients and normal subjects We assessed 847 human miRNA in the microarray. Fold changes greater than 2 or less than ⫺2 were considered significant. Moreover, a P value threshold ⬍0.05 was set for statistical significance. As shown in Fig. 1A, we found 16 miRNA had significantly different expression in GD PBMC compared with control PBMC. Among them, 14 miRNA were suppressed, whereas two miRNA were enhanced in GD PBMC compared with normal PBMC.

The direct effect of T3 on the expression of miRNA in PBMC To better characterize the relationship between the selective miRNA and GD as well as evaluate the effect of thyroid hormone on miRNA regulation in immune cells, we also examined expression levels of the miRNA in PBMC directly under T3 treatment. We isolated fresh PBMC from healthy individuals and cultured them with or without T3 treatment. After both 24 h and 7 d of cell culture, we found that miR-154*, miR-376b, and miR431* were all significantly down-regulated in PBMC with T3 treatment compared with the groups without T3 (Fig. 1, C and D). Our results revealed that all three selected miRNA could be suppressed directly by acute and chronic T3 exposure in PBMC in vitro.

Validation of the miRNA expression Three top-ranked miRNA, miR-154*, miR-376b, and miR-431*, were selected for qRT-PCR validation based

miRNA targets and biological pathways prediction To understand the possible functions of the selective miRNA, we used two bioinformatics prediction databas-

FIG. 1. Differential miRNA expressions in PBMC from GD patients compared with normal controls. A, Microarray data showing significantly changed miRNA in PBMC from GD patients compared with normal subjects; B, differential miR-154*, miR-376b, and miR-431* expression verified by qRT-PCR in PBMC from healthy controls, GD patients, and GD patients in remission; C and D, expression of miR-154*, miR-376b, and miR431* in freshly cultured healthy human PBMC after 24 h and 7d with or without T3 treatment.


es: MicroCosm Targets V5 and Targetsan (targetscan custom for miRNA*) for putative target genes prediction. Supplemental Tables 2 and 3 list the numbers and names of predicted target genes for miR-154* and miR-376b according to each database. We examined these genes in PBMC and found that four genes were significantly elevated in GD patients and repressed after methimazole treatment and might be potential target genes (Supplemental Table 4 and Supplemental Fig. 1). However, we didn’t get any common predicted target genes for miR431*, which indicated that its function could be very novel. The PANTHER database classifies genes into families and subfamilies by their functions and identifies pathways that are enriched in the list of miRNA target genes. We used this database to categorize predicted target genes of miR-154* and miR-376b (Supplemental Table 5). The genes targeted by these miRNA were involved in classical immunological pathways, such as chemokine and cytokine signaling pathways, and also in the G protein pathway and Wnt signaling pathway. These predicted pathways might provide us with more clues to study the molecular mechanisms for GD pathogenesis.

Discussion In this study, by systematically screening and verifying three differentially expressed PBMC miRNA in GD patients and healthy controls, we revealed the significance of miRNA on GD for the first time. miR-154*, miR-376b, and miR-431* were confirmed to be significantly suppressed in GD patients compared with normal controls. Meanwhile, the expression of these three miRNA in primary PBMC culture could be suppressed directly by T3 treatment. T3 and T4 could regulate innate and adaptive immune responses through genomic and nongenomic mechanisms (11). In this study, we provided a novel possibility that T3 might modulate the immune system via miRNA. Software prediction showed that these miRNA were involved in various chemokine and cytokine signaling pathways. Therefore, the down-regulation of these miRNA in GD might evoke proinflammatory and immunological responses and exacerbate GD development. In addition, these selective miRNA might have a regulatory function on, e.g. the G protein pathway and Wnt signaling pathway. These pathways have recently been shown to be involved in GD pathogenesis. For instance, The T393C polymorphism of the G protein ␣s gene is associated with the course of GD (12). In addition, genes of the Wnt pathway were recently reported to be up-regulated in chronic


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Graves’ ophthalmopathy (13). Moreover, we examined predicted target genes and found four potential candidates. HAS2, for instance, was responsible for synthesizing hyaluronic acid, which is a major ligand for adhesion receptor CD44 and associated with inflammatory response (14). However, the functions of these genes in GD still need to be studied. We also noticed an interesting phenomenon that in the microarray data, nine of 16 significantly changed miRNA in GD patients were star-form miRNA (miRNA*). Furthermore, among the three top-ranked miRNA from the microarray data, two were star-forms (miR-154* and miR-431*). Generally, the mature miRNA is defined as the duplex strand that accumulates to a high steady-state level. However, its partner strand, called the miRNA* or passenger strand, was considered as an inactive strand and generally degraded (15). But this point of view has been challenged in some recent studies. By analyzing miRNA/ miRNA* sequences across vertebrates, Guo et al. (16) had a conclusion that well-conserved miRNA* strands, particularly conservation in seed sequences may afford potential opportunities for a regulation network. Okamura et al. (17) found that Drosophila melanogaster miRNA* species are often present at physiologically relevant levels and capable of repressing targets in both cultured cells and animals. Meanwhile, it is reported that miR-9 and miR-9* could target the transcription factor repressor element-1 silencing transcription factor and corepressor for element1-silencing transcription factor, respectively, in Huntington’s disease (18). Zhou et al. (4) found that miR-155 and miR-155* can regulate type I interferon production by human plasmacytoid dendritic cells cooperatively and had opposite effects. All these studies suggested the importance of miRNA* in biological processes and disease physiology. Our present study supports such a notion, indicating that miRNA* was also involved in GD pathogenesis. In summary, we got consistent data showing that the PBMC expressions of miR-154*, miR-376b, and miR431* were suppressed in GD patients and recovered in GD patients in remission. Meanwhile, T3 treatment could directly inhibit these miRNA expressions in cultured PBMC. Our findings revealed the differentially expressed miRNA associated with GD, which might serve as novel biomarkers of GD and potential targets for GD treatment.

Acknowledgments We thank Xiaoying Li, Yichen Qi, and Xiaoli Li (Department of Endocrinology and Metabolism, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine) for assistance in preparing and submitting the manuscript.

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Address all correspondence and requests for reprints to: Dongping Lin, Department of Endocrinology and Metabolism, the Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China. E-mail: [email protected]; or Shu Wang, Shanghai Clinical Center for Endocrine and Metabolic Diseases, and Department of Endocrine and Metabolic Diseases, Rui-Jin Hospital, Affiliated to Shanghai Jiao-Tong University School of Medicine, 197 Rui-Jin Second Road, Shanghai 200025, China. E-mail: [email protected]. This study was supported by grants from the National Natural Science Foundation of China (30725037 and 30971385) and Science and Technology Commission of Shanghai Municipality (09ZR1416100). Disclosure Summary: The authors have no duality of interest to declare.


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