Abstract
The aim of this study is to characterize the microRNA (miRNA) expression signatures in patients with thyroid eye disease (TED) and identify miRNA biomarkers of disease activity. Total RNA was isolated from the sera of patients with TED (n = 10) and healthy controls (HCs, n = 5) using the miRNeasy Serum/Plasma Kit. The NanoString assay was used for the comprehensive analysis of 798 miRNA expression profiles. Analysis of specific miRNA signatures, mRNA target pathway analysis, and network analysis were performed. Patients with TED were divided into two groups according to disease activity: active and inactive TED groups. Differentially expressed circulating miRNAs were identified and tested using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) tests in the validation cohort. Among the 798 miRNAs analyzed, 173 differentially downregulated miRNAs were identified in TED patients compared to those in the HCs. Ten circulating miRNAs were differentially expressed between the active and inactive TED groups and regarded as candidate biomarkers for TED activity (one upregulated miRNA: miR-29c-3p; nine downregulated miRNAs: miR-4286, miR-941, miR-571, miR-129-2-3p, miR-484, miR-192-5p, miR-502-3p, miR-597-5p, and miR-296-3p). In the validation cohort, miR-484 and miR-192-5p showed significantly lower expression in the active TED group than in the inactive TED group. In conclusion, the expression levels of miR-484 and miR-192-5p differed significantly between the active and inactive TED groups, suggesting that these miRNAs could serve as circulating biomarkers of TED activity, however, these findings need to be validated in further studies.
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Introduction
Thyroid eye disease (TED) is the most common orbital inflammatory disease, and extrathyroidal manifestation of Grave’s disease (GD)1. Most patients with TED show distinct clinical features, including eyelid retraction, proptosis, extraocular muscle movement limitation, and inflammatory signs of eyelid and conjunctiva, which enable easy diagnosis. Although enormous progress has been made in elucidating the pathogenesis of TED, the exact mechanism of TED remains elusive, and therapeutic challenges and dilemmas remain.
Assessment of disease activity is crucial for designing treatment plans for patients with TED. Immunosuppressive agents, including corticosteroids, radiation treatment, and insulin-like growth factor-1 receptor (IGF-1R) antibodies are effective only in patients with active TED2,3,4. Surgical rehabilitation is the conventional treatment modality for inactive TED. The clinical activity score (CAS) is the most commonly used to measure the activity of TED in a clinical setting5. However, CAS contains subjective items such as spontaneous retrobulbar pain and pain on attempted eye movement. Furthermore, fine, quantitative analysis is not possible because each item of the CAS is binary6. These drawbacks warrant the development of novel biomarkers for measuring TED activity.
MicroRNAs (miRNAs) are a class of noncoding RNAs that post-transcriptionally regulate gene expression and are involved in a wide range of physiological and pathological processes36 investigated the association between the changes in levels of miR-146a and IL-7 with the clinical activity of TED using qRT-PCR and ELISA and reported lower miR-146a levels and higher IL-7 levels in active TED than in inactive TED. Shen et al.33 previously evaluated the level of circulating miRNAs according to the steroid responsiveness in TED patients. They used miScript PCR arrays for screening and qPCR for validation, like in case of the present study. However, they only included patients with active TED and compared the level of 84 miRNAs between steroid responders and non-responders. The strength of this study is that we performed the analysis of the expression of a wide panel of miRNAs. Here, we explored the levels of 798 miRNAs and used a very sensitive method to detect miRNAs in serum.
This study has some limitations. First, the sample size was limited; thus, the results from this study should ideally be validated in a much larger study population. Furthermore, the exact function of miR-484 and miR-192-5p in the pathogenesis of TED remains unclear, and in vitro or in vivo experimental studies regarding these miRNAs are warranted. Second, heterogeneous demographic, endocrinologic, and ophthalmologic factors can affect the expression of circulating miRNAs. Therefore, we plan to conduct further studies on the comparative analysis of miRNA expression in sequential serum samples collected from the same patient. Third, we performed only traditional biological experiments involving the screening of a broad panel of miRNAs, and validation of candidate miRNAs. Recently, there is a trend of develo** computational models to identify the associations between miRNAs and various human diseases37,38,39. These computational models can effectively integrate heterogeneous biological data and predict potential associations between miRNAs and human diseases. Future studies are expected to develop a computational model that can predict miRNAs related to TED. Forth, we identified 10 miRNAs as potential biomarkers of TED activity using the NanoString nCounter system, but we successfully amplified only 6 of 10 miRNAs using qRT-PCR. We speculated that miRNAs that exist in scarce amounts in the serum might fail to be amplified by qRT-PCR, although they were detected using an amplification-free technique.
In conclusion, we investigated the expression of a wide panel of 798 miRNAs in patients with TED and identified 10 miRNAs which were differentially expressed between active and inactive TED. Validation studies revealed that the serum levels of miR-484 and miR-192-5p were significantly lower in active TED than in inactive TED. These miRNAs have the potential to serve as serum biomarkers of disease activity in TED. However, studies with larger sample sizes are needed to verify the roles of these miRNAs in the pathogenesis of TED.
Data availability
The datasets generated and analyzed during the present study are available in the Gene Expression Omnibus repository (GSE207995).
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Funding
This research was supported by the National Research Foundation (NRF) grant funded by the Korea government (No. 2019R1G1A1100257). The funding organization had no role in the design or conduct of this research.
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N.K., H-K.C, S.I.K and M.J.L conceived and designed the study. S.E.W and M.K.Y. collected the data, analyzed the experimental data. Y.J.K and S.E.W. did part of the experiments. All authors designed the work, read and approved the final manuscript.
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Kim, N., Choung, H., Kim, Y.J. et al. Serum microRNA as a potential biomarker for the activity of thyroid eye disease. Sci Rep 13, 234 (2023). https://doi.org/10.1038/s41598-023-27483-w
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DOI: https://doi.org/10.1038/s41598-023-27483-w
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