Department of Ophthalmology, Eye & ENT Hospital, Shanghai Medical College, Fudan University, 83 Fenyang Road, Shanghai, China; NHC Key Laboratory of Myopia, Fudan University, 83 Fenyang Road, Shanghai, China; Key Laboratory of Myopia, Ministry of Health, Fudan University, 83 Fenyang Road, Shanghai, China.
Department of Ophthalmology, First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, Zhejiang, China.
Exp Eye Res. 2020 Apr;193:107962. doi: 10.1016/j.exer.2020.107962. Epub 2020 Feb 10.
Our study aimed to reveal the underlying pathologic mechanisms of thyroid-associated ophthalmopathy (TAO) by integrative transcriptomics and proteomic analysis of extraocular muscles (EOM). The study involved 11 TAO patients (clinical activity score ≤ 2) and 11 control donors. Total RNA was extracted from EOM samples of 5 TAO patients and 5 control individuals for gene microarray analysis to reveal differentially expressed genes. Concurrently, EOM samples from 3 TAO patients and 3 control individuals were lysed for quantitative proteomic analysis. Differentially expressed genes and proteins were identified, followed by functional and pathway enrichment analysis and protein-protein interaction network construction. Concordance between proteins and transcripts was examined, and functional annotations were conducted. Expressions of versican (VCAN) and lipocalin 1 (LCN1) in EOM samples from another 3 TAO patients and 3 control individuals were measured by western blotting. In total, 952 genes and 137 proteins were identified as differentially expressed, as well as 96 differentially expressed proteins without significantly changed mRNA abundance. Proteins mainly related to the composition (such as MYH1, MYH2, and MYH13) and contraction force (MYH3, MYH8, ACTN3, and TNNT1) of the muscle fibers were significantly up-regulated in EOM samples of TAO, as well as those (such as VCAN, MPZ, and PTPRC) associated with cell adhesion. In addition, differentially expressed proteins related to the components and metabolism of extracellular matrix (ECM) (such as COL1A1, COL1A2, COL2A1, VCAN, OGN, and DCN) were identified. Similarly, expressions of genes involved in cell adhesion and ECM metabolism were significantly different between EOM samples of TAO patients and controls. Western blotting verified that VCAN involved in ECM proteoglycans and diseases associated with glycosaminoglycan metabolism was markedly higher in EOM samples of TAO, whereas LCN1 was obviously decreased. In conclusion, this study demonstrated the significantly altered cellular components of EOM, muscle contraction, cell adhesion and ECM metabolism, which might be involved in the pathologic mechanisms and/or consequences of TAO.
我们的研究旨在通过对眼外肌（EOM）的整合转录组学和蛋白质组学分析，揭示甲状腺相关眼病（TAO）的潜在病理机制。该研究纳入了 11 名 TAO 患者（临床活动评分≤2）和 11 名对照供体。从 5 名 TAO 患者和 5 名对照个体的 EOM 样本中提取总 RNA，进行基因微阵列分析以揭示差异表达基因。同时，裂解 3 名 TAO 患者和 3 名对照个体的 EOM 样本进行定量蛋白质组学分析。鉴定差异表达基因和蛋白质，然后进行功能和途径富集分析以及蛋白质-蛋白质相互作用网络构建。检查蛋白质和转录物之间的一致性，并进行功能注释。通过 Western blot 测量另 3 名 TAO 患者和 3 名对照个体的 EOM 样本中的神经蛋白聚糖（VCAN）和脂钙蛋白 1（LCN1）的表达。总共鉴定出 952 个基因和 137 种蛋白质作为差异表达，以及 96 种差异表达的蛋白质，其 mRNA 丰度没有明显变化。蛋白质主要与肌肉纤维的组成（如 MYH1、MYH2 和 MYH13）和收缩力（如 MYH3、MYH8、ACTN3 和 TNNT1）相关，与细胞粘附相关的蛋白质（如 VCAN、MPZ 和 PTPRC）也显著上调。此外，还鉴定出与细胞外基质（ECM）的组成和代谢相关的差异表达蛋白（如 COL1A1、COL1A2、COL2A1、VCAN、OGN 和 DCN）。同样，TAO 患者和对照 EOM 样本之间涉及细胞粘附和 ECM 代谢的基因表达也有显著差异。Western blot 验证了 EOM 样本中涉及 ECM 蛋白聚糖和糖胺聚糖代谢的 TAO 相关基因 VCAN 明显升高，而 LCN1 明显降低。总之，这项研究表明 EOM 的细胞成分、肌肉收缩、细胞粘附和 ECM 代谢发生了显著改变，这些改变可能与 TAO 的病理机制和/或后果有关。
