Integrative transcriptomics and proteomic analysis of extraocular muscles from patients with thyroid-associated ophthalmopathy

Journal Pre-proof Integrative transcriptomics and proteomic analysis of extraocular muscles from patients with thyroid-associated ophthalmopathy Lianqun Wu, Shujie Zhang, Xiuyi Li, Jing Yao, Ling Ling, Xiao Huang, Chunchun Hu, Yihan Zhang, Xiantao Sun, Bing Qin, Guohua Liu, Chen Zhao PII:

S0014-4835(19)30577-9

DOI:

https://doi.org/10.1016/j.exer.2020.107962

Reference:

YEXER 107962

To appear in:

Experimental Eye Research

Received Date: 18 August 2019 Revised Date:

9 January 2020

Accepted Date: 5 February 2020

Please cite this article as: Wu, L., Zhang, S., Li, X., Yao, J., Ling, L., Huang, X., Hu, C., Zhang, Y., Sun, X., Qin, B., Liu, G., Zhao, C., Integrative transcriptomics and proteomic analysis of extraocular muscles from patients with thyroid-associated ophthalmopathy, Experimental Eye Research (2020), doi: https:// doi.org/10.1016/j.exer.2020.107962. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Integrative transcriptomics and proteomic analysis of extraocular muscles from patients with thyroid-associated ophthalmopathy Lianqun Wua,b,c,1, Shujie Zhanga,b,c,1, Xiuyi Lid, Jing Yaoa,b,c, Ling Linga,b,c, Xiao Huange, Chunchun Hua,b,c, Yihan Zhanga,b,c, Xiantao Sunf, Bing Qing, Guohua Liuh, Chen Zhaoa,b,c* a

Department of Ophthalmology, Eye & ENT Hospital, Shanghai Medical College, Fudan University, 83 Fenyang Road, Shanghai, China.

b c

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.

d

Department of Ophthalmology, First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou,

Zhejiang, China. e

Department of Ophthalmology, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai, China.

f

Department of Ophthalmolgoy, Henan Children’s Hospital, 255 Gangdu Road, Zhengzhou, China.

g

Department of Ophthalmolgoy, Suqian First Hospital, 120 Suzhi Road, Suqian, China.

h

Department of Ophthalmology, Qilu Children’s Hospital of Shandong University, 430 Jingshi Road, Jinan, China.

1

Joint first authors.

*Corresponding author: Chen Zhao, Department of Ophthalmology, Eye & ENT Hospital of Fudan University, 83 Fenyang Road, Shanghai, 200031, China. E-mail address: [email protected]. Declarations of interest: none.

Abstract 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.

Keywords: thyroid-associated ophthalmopathy, gene microarray, proteomic profiling, extraocular muscle

1.

Introduction Thyroid-associated ophthalmopathy (TAO) is a process in which orbital connective tissues and extraocular muscles (EOM) undergo

inflammation and remodeling resulting from autoimmune responses to antigens shared by the thyroid and orbit, most commonly occurring in Graves’ disease (Romero-Kusabara et al., 2017; Smith, 2019a). Clinical manifestations of TAO are mainly proptosis, eyelid retraction, edema, eye movement limitation with diplopia, redness of eyelid, periorbital tissues and conjunctiva (Bahn, 2010). Better understanding of pathologic mechanisms of TAO could contribute to improved etiologic treatments of TAO. Thyroid stimulating hormone receptor (TSHR) is recognized as the primary autoantigen of Graves’ disease, which is diagnosed according to the circulating auto-antibodies against TSHR in a patient’s blood (Menconi et al., 2014). Levels of TSHR mRNA are increased in orbital fibroblasts (OFs) from patients with TAO, compared to those from normal controls (Douglas et al., 2010). Insulin-like growth factor-1 receptor (IGF-1R), a transmembrane tyrosine kinase receptor, can form both physical and functional complexes with the TSHR (Smith, 2019b). Inhibition of IGF-1R activity by monoclonal antibodies could suppress the downstream signaling of TSHR, suggesting that blocking the IGF-1R pathway might be a useful strategy for TAO therapy (Tsui et al., 2008). A number of pro-inflammatory cytokines have been identified as potentially associated with the pathogenesis of TAO (Smith, 2002). It has been unraveled that unusual susceptibility of OFs to pro-inflammatory cytokines can contribute to cytokine-dependent fibroblast activation, leading to remodeling of EOM and orbital connective tissues (Smith, 2005). In the study of Hiromatsu et al., the enlargement of EOM was found significantly correlated with mRNA expression of tumor necrosis factor (TNF)-α in EOM, and the orbital volume was positively correlated with interleukin (IL)-6 mRNA expression and negatively correlated with IL-4 mRNA and IL-10 mRNA expression in orbital fat tissue (Hiromatsu et al., 2000). OFs that are activated by the infiltrated lymphocytes produce glycosaminoglycans and differentiate into myofibroblasts or adipocytes, leading to proptosis and compression of the optic nerve (Kumar

et al., 2004). Although progress has been made in understanding the pathological mechanism of TAO, the exact pathogenesis of TAO involving complex molecules and processes is not yet fully understood. The goal of our study was to reveal more underlying pathologic mechanisms of TAO by integrative transcriptomics and proteomic analysis of EOM from TAO patients. EOM samples of TAO patients and control donors were used for gene microarray analysis and quantitative proteomic analysis. The correlations between the proteins and transcripts were determined. In addition, protein expression changes of versican (VCAN) and lipocalin 1 (LCN1) in EOM samples of TAO patients were verified by western blotting.

2.

Materials and methods

2.1. Subjects and tissue sample Our study enrolled 11 TAO patients from the Department of Ophthalmology, Changzheng Hospital. TAO was diagnosed according to the Bartley criteria (Bartley and Gorman, 1995). The activity of TAO was assessed using the 7-point modified formulation of the clinical activity score (CAS), which consists of 7 classic signs of inflammation including spontaneous pain, pain during eye movement, redness of the conjunctiva, redness of the eyelids, swelling of caruncle or plica, swelling of eyelids, and swelling of conjunctiva (chemosis) (Mourits et al., 1997). The score ranges from 0 to 7, where CAS ≥ 3 was defined as active TAO, and CAS < 3 was defined as inactive TAO (Huang et al., 2012). Patients were excluded from the study if they met one of the following conditions: received radioactive iodine therapy or thyroidectomy; suffered from other ocular or systemic inflammatory or autoimmune diseases; received systemic or local steroids and/or immunosuppressive agents within 6 months. All the recruited TAO patients were hyperthyroid upon initial diagnosis of TAO, with a CAS of less than 3 for at least 6 months. Their thyroid functions recovered to normal range after antithyroid drug treatments. EOM samples of the 11 TAO patients were obtained from the waste during strabismus surgery, and 11

normal EOM samples of control individuals were from deceased organ donors under the supervision of the institutional review board. All the participants were Chinese, and EOM samples were lateral rectus muscles. The demographic results and clinical profiles of TAO patients and controls were summarized in supplementary table1. All the controls were without history of strabismus, thyroid disease, or orbital inflammatory conditions. This study was approved by the Ethics Committee of Changzheng Hospital (2018SL039A), which is affiliated to the Second Military Medical University. Written informed consent was obtained from all the patients, and the research protocol was performed in compliance with the tenets of the Declaration of Helsinki. 2.2. RNA isolation, labeling, hybridization, and scanning Total RNA was extracted from 5 EOM samples of TAO patients and 5 EOM samples from control donors using TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. Sample labeling and array hybridization were performed in accordance with the modified Agilent One Color Microarray Based Gene Expression Analysis protocol (Agilent Technology) previously described (Guo et al., 2018). In brief, each purified mRNA was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3' bias by a random priming method. Arraystar Human LncRNA Microarray V4.0 was employed to hybridize the labeled cRNAs. Hybridization solution was dispensed into the gasket slide and assembled with the lncRNA expression microarray slide. The slides were incubated for 17 h at 65 °C in an Agilent Hybridization Oven and scanned with the Agilent DNA Microarray Scanner (part number G2505C). 2.3. Gene microarray analysis Microarray expression data were normalized using the Limma package, including background correction and quantile normalization. Differentially expressed genes were identified by t test, with the cutoff criteria of p value < 0.05 and |log2FC (fold change)| > 1. Concurrently, hierarchical clustering analysis was conducted for differentially expressed genes by the R package “pheatmap” based on

gene expressions in EOM samples of 5 TAO patients and 5 control donors. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for differentially expressed genes was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID, v6.8) (Huang et al., 2009) with the threshold of p value < 0.05. 2.4. Protein extraction EOM samples from 3 TAO patients and 3 control donors were lysed using sodium dodecyl sulfate (SDS) lysis buffer (Beyotime, China) supplemented with 1mM phenylmethanesulfonyl fluoride (PMSF; Amresco). Samples were centrifuged twice at 15,000g for 15 min after sonication. The supernatants were kept at -20 °C overnight. Protein concentration was determined by a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Pierce Biotechnology, USA) and aliquot samples were stored at -80 °C. Each protein sample (15 µg) was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were stained with Coomassie brilliant blue (CBB) G-250 (Sigma) according to Candiano’s protocol (Candiano et al., 2004). Stained gels were scanned by ImageScanner (GE Healthcare, USA) at a resolution of 300 dots per inch. 2.5. Trypsin digestion and TMT labeling Each protein sample (100 µg) was used for peptide extraction with trypsin by filter-aided sample preparation (FASP) digestion with some modifications (Wiśniewski et al., 2009). Briefly, proteins in the 10 kDa cut-off spin filter were reduced by 10 mM dithiothreitol (DTT) in 8 M urea/0.1 M triethylammonium bicarbonate (TEAB) (pH 8.0) at 60 °C for 1 h and subsequently reacted with iodoacetamide (IAA; 50 mM) for 40 min in the dark. The solution was then concentrated by centrifugation at 12,000 rpm for 20 min. Afterwards, 100 µl of 0.3M TEAB was added into the filter containing the reduced proteins and centrifuged at 12,000 rpm for 20 min. This step was repeated twice. Proteins were digested with trypsin (2 µg; Beijing Hualishi Technology Co., Ltd, China) at 37 °C for 12 h. Spin filter

containing peptides was centrifuged at 12,000 rpm for 20 min to collect the peptides. Then, the collected peptides were lyophilized using a freeze dryer (Ningbo Scientz Biotechnology Co., Ltd, Ningbo, China). The dried peptides were resuspended with 100 µl of 0.2M TEAB and labeled with the TMT using a 6-plex TMT kit (Thermo Fisher Scientific, Waltham, MA, USA). Each peptide solution (40 µl) was added with 41 µl of anhydrous acetonitrile and 41 µl of TMT label reagent, and the reaction mixtures were incubated at room temperature for 1 h. The reactions were quenched with 8 µl of 5% hydroxylamine for 15 min at room temperature. The derivatized peptides were lyophilized and stored at -80 °C. 2.6. Quantitative proteomic analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) Samples were fractionated by reverse-phase high-performance liquid chromatography (HPLC) on an Agilent 1100 Series HPLC system (Agilent Technologies), according to the protocol described previously (Plubell et al., 2017). We used an Agilent Zorbax Extend C18 column (5 µm particle size, 150 mm × 2.1 mm) with a flow rate of 300 µl/min. A 75 min gradient using solvent A (2% acetonitrile in H2O) and solvent B (90% acetonitrile in H2O) was run as follows: 0~8 min, 98% A; 8~8.01 min, 98%~95% A; 8.01~48 min, 95%~75% A; 48~60 min, 75~60% A; 60~60.01 min, 60~10% A; 60.01~70 min, 10% A; 70~70.01 min, 10~98% A; 70.01~75 min, 98% A. Ultraviolet (UV) absorbance was measured throughout the gradient at 210 nm and 280 nm. Fractions were harvested from 8 min to 60 min and concatenated into 15 samples by combining multiple fractions. Samples were lyophilized for mass spectrometry (MS) detection. Samples were dissolved in 0.1% formic acid (FA) and directly loaded onto a reversed-phase precolumn (Acclaim PepMap 100, 100 µm × 2 cm, Thermo Scientific, USA). Peptide separation was performed using a reversed-phase analytical column (Acclaim PepMap RSLC, 75 µm × 15 cm, Thermo Scientific, USA). The gradient was comprised of increasing solvent B (0.1% FA, 80% ACN, 19.9% H2O) in solvent A (0.1% FA, 99.9% H2O) from 0% to 20% over 32 min, 20% to 40% over 15 min and 40% to 90% over 3 min, at a constant flow rate of 300 nl/min on a Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific, USA).

Peptides were analyzed with a Q Exactive HF MS instrument (Thermo Fisher Scientific, USA). Acquisition resolution was set to 120,000 with an automatic gain control (AGC) target value of 3e6 and maximum ion injection time of 50 ms for a scan range of 300– 1500 m/z. The top 12 most intense peaks in MS were fragmented with higher-energy collisional dissociation with normalized collisional energy (NCE) of 32, ion fragments were detected at a resolution of 45,000 with an AGC target value of 1e5 and a max injection time of 100 ms. The Q-Exactive HF dynamic exclusion was set for 30.0 s and run under positive mode. 2.7. Proteomics data analysis All LC-MS/MS raw files were analyzed using Proteome Discoverer 2.2 (Thermo Fisher Scientific, USA). The protein annotation data were searched against the protein sequence database of Homo sapiens from the Uniprot database. All confidently identified peptides with SEQUEST-HT scores > 0 and proteins identified by ≥ 1 unique peptide remained. False discovery rate (FDR) thresholds for proteins, peptides, and modification sites were set as 1%. Differentially expressed proteins were identified by t test, with the cutoff criteria of p value < 0.05 and |log2FC| > 1. Principle component analysis (PCA) of the EOM samples from 3 TAO patients and 3 control individuals was performed. Meanwhile, we performed a hierarchical clustering of differentially expressed proteins by the R package “pheatmap” based on expressions in EOM samples of 3 TAO patients and 3 control individuals. GO annotation and KEGG pathway enrichment analysis for differentially expressed proteins was performed using the DAVID (v6.8) (Huang et al., 2009) with the threshold of p value < 0.05. Gene set enrichment analysis (GSEA) uses a weighted Kolmogorov-Smirnov-like statistic to calculate the pathway enrichment score, ranking all genes by their significance levels of enrichment scores and testing the distribution of each pathway’s member in the entire ranked list (Subramanian et al., 2005). For further interpreting expression data, GSEA (http://www.broadinstitute.org/gsea/) was applied to determine sets of genes from the enriched

KEGG pathways. Protein-protein interaction (PPI) networks for up-regulated and down-regulated differentially expressed proteins were also conducted by using the search tool for the retrieval of interacting genes (STRING; http://string-db.org) (Szklarczyk et al., 2016). The minimum required interaction score was set to 0.4. 2.8. Integrative transcriptomics and proteomic analysis mRNA expression and protein quantification data were annotated to gene symbols, respectively. We examined the concordance between proteins and transcripts, and correlation analysis was performed. In addition, functional annotations were conducted for differentially expressed proteins without mRNA transcript alteration. 2.9. Verification by western blotting The expressions of LCN1 and VCAN in EOM samples from 3 TAO patients and 3 control donors were measured by western blotting. Briefly, protein extracted from each sample was separated by SDS-PAGE and transferred onto nitrocellulose (NC) membranes. After blocking in TBST buffer containing 5% BSA for 1 h at 37 °C, membranes were probed with antibodies of LCN1 (1:1000; GeneTex, GTX114523), VCAN (1:1000; Abcam, ab177480), and GAPDH (1:1000; Sangon, D110016) at 4 °C overnight. Membranes were washed 3 times with TBST for 5 min, followed by incubation with HRP-conjugated secondary antibodies for 1 h, and then incubated with chemiluminescent substrate. Images were captured using an image analyzer LAS-4000 mini (GE Healthcare).

3.

Results

3.1. Differentially expressed genes in TAO samples and enrichment analysis In total, 15,255 genes were identified in the EOM samples. Using the cutoff criteria of P value < 0.05 and |log2FC| > 1, we identified

952 differentially expressed genes, including 304 up-regulated and 648 down-regulated genes (Figure 1A). A hierarchical clustering heatmap showed that these 952 differentially expressed genes had obvious different expression patterns between normal control samples and TAO samples (Figure 1B). The identified differentially expressed genes were significantly related to the neutrophil chemotaxis (such as CCL23, CCL14, and C5AR1), cell adhesion (such as ATP1B2, TNC, and ITGA11), UDP-N-acetylglucosamine biosynthetic process (such as RENBP, UAP1, and GNPDA1), defense response to fungus (such as S100A8, S100A9, and DEFA4), chemotaxis (such as RNASE2, C5AR1, and CMTM2), and immune response (such as TNFRSF6B, ENPP2, CXCL3, and IL18) (Supplementary table 2, Supplementary figure 1). Meanwhile, KEGG pathways were enriched for these differentially expressed genes, including salivary secretion (such as ADCY2, ADCY7, and PRH2), extracellular matrix (ECM)-receptor interaction (such as COL4A3, CD36, and COL6A6), amino sugar and nucleotide sugar metabolism (such as RENBP, UAP1, and GNPDA1), cytokine-cytokine receptor interaction (such as IL17RA, TNFRSF10C, CCL23, and CCL14), complement and coagulation cascades (such as PLAT, CD55, and F10) (Supplementary table 3, Supplementary figure 2). 3.2. Differentially expressed proteins associated with TAO There were 3,866 confidently identified proteins, among which 137 proteins were identified as differentially expressed proteins (41 up-regulated and 96 down-regulated) in the EOM samples of TAO patients (Figure 2A). The protein expression levels of MZB1, MYH8, CSRP3, MYH13, IKBKB, CD53, NAIF1, RPS4Y1, MYH1, MYH4, SMTNL1, ANK1, MPZ, SERPINE2 and VCAN were the top 15 significantly up-regulated proteins in the EOM samples of TAO patients, whereas PTPRN2, COL8A2, KRT4, COL11A2, MMP10, LCN1, ALDH3A1, KRT13, KRT5, ADH7, PAWR, ANGPTL7, SORBS2, COL2A1, and SFN were the top 15 significantly down-regulated proteins in EOM samples of TAO patients (Table 1).

Two-dimensional scatter plots of the principal components of the EOM samples showed a good clear separation between samples from TAO and control patients (Figure 2B). A hierarchical clustering heatmap showed that, based on the expression pattern of the 137 differentially expressed proteins, samples from TAO and control groups formed two distinct separate clusters, suggesting differentially expressed proteins could well distinguish normal control samples from TAO samples (Figure 2C). 3.3. Enrichment analysis of differentially expressed proteins In order to reveal the potential roles of differentially expressed proteins, GO annotation and KEGG pathway enrichment were applied for the differentially expressed proteins. GO annotation revealed that many differentially expressed proteins (such as COL1A1, COL1A2, COL2A1, COL11A2, DPT, and FMOD) were related to core matrisome and supramolecular fiber organization (Figure 3A). Differentially expressed proteins, including VCAN, DCN, FMOD, OGN, PRELP, and KERA, were associated with proteoglycans. Humoral immune response and cell recognition were involved with IGHD, IGHV6-1, VCAN, and IGHV3-43. Further, IGLC7, CST4, LCN1, and LTF were related to retina homeostasis and negative regulation of hydrolase activity (Table 2). Pathway enrichment analysis indicated that differentially expressed proteins were significantly involved in 12 KEGG pathways, such as tight junction, protein digestion and absorption, focal adhesion, and ECM-receptor interaction (Figure 3B, Table 3). GSEA showed that tight junction was mostly associated with up-regulated proteins (such as MYH1, MYH4, MYH6, MYH8, MYH3, MYH13 and ACTN3), and pathway of cell adhesion molecules was also related to up-regulated proteins (such as VCAN, MPZ, and PTPRC) (Figure 3C, Table 3). On the contrary, ECM-receptor interaction was highly associated with down-regulated proteins (such as COL1A1, COL1A2, COL6A6, and COL2A1). 3.4. PPI networks Based on the PPI data from STRING, PPI networks for up-regulated and down-regulated differentially expressed proteins were

constructed. There were five up-regulated proteins that connected with ≥ 4 proteins, including MYH1 (node = 9), MYH4 (node = 7), MYOM2 (node = 7), MYH6 (node = 5), and MYH8 (node = 5) (Figure 4A). VCAN was interacted with POSTN in the PPI network of up-regulated proteins. In the PPI network for down-regulated proteins, the top five hub proteins were KRT14 (node = 14), COL1A1 (node = 13), KRT13 (node = 10), KRT5 (node = 10) and DCN (node = 9) (Figure 4B). LCN1 was interacted with eight proteins, including PIP, LYZ, KERA, SCGB1D1, CST4, LTF, SCGB2A1, and LACRT. 3.5. Integrative transcriptomics and proteomic analysis In total, 96 differentially expressed proteins (26 up-regulated and 70 down-regulated proteins) without significantly changed mRNA abundance were observed by a combination of protein quantification data and mRNA expression data (Figure 5A). Functional annotations were conducted for these 96 differentially expressed proteins without mRNA transcript alteration. Among the revealed functions, differentially expressed proteins without mRNA transcript alteration were mainly related to cellular components of EOM (such as collagen fibrils and ECM), striated muscle contraction (MYH3, MYH8, ACTN3, and TNNT1), and ECM proteoglycans and glycosaminoglycan metabolism. VCAN was involved in ECM proteoglycans and diseases associated with glycosaminoglycan metabolism, whereas COL1A1, COL1A2, and COL2A1 were related to MET promotes cell motility, collagen formation, MET activates PTK2 signaling, and degradation of the ECM (Figure 5B). 3.6. Differential expressions of VCAN and LCN1 According to the results of western blotting, protein levels of VCAN were markedly higher in EOM samples collected from TAO patients; LCN1 was obviously decreased in TAO samples compared with the control sample (Figure 6). These results were consistent with those of the proteomic analysis.

4.

Discussion TAO involves orbital and extraocular tissue; however, intraocular complications such as elevated intraocular pressure, optic papilla

edema, and choroidal folds may occur as well, even leading to loss of vision (Wiersinga et al., 2006). Currently, the medical and surgical therapies for TAO are inadequate, largely due to our incomplete understanding of disease pathogenesis. Here, an integrative analysis of proteomic alterations and transcriptomics data derived from EOM of TAO patients and control donors was performed. Gene microarray analysis for 5 EOM samples of TAO patients and 5 EOM samples from control donors revealed 952 differentially expressed genes. Functional enrichment analysis showed these differentially expressed genes were mainly related to neutrophil chemotaxis, cell adhesion, UDP-N-acetylglucosamine biosynthetic process, defense response to fungus, and immune response. Besides, differentially expressed genes were significantly enriched in salivary secretion, ECM-receptor interaction, amino sugar and nucleotide sugar metabolism, cytokine-cytokine receptor interaction, complement and coagulation cascades. According to the tissue metabolite profiling analysis, sugar metabolism, as well as glycerolipid and amino-sugar metabolism were found significantly altered in Graves’ ophthalmopathy (Ji et al., 2018). Meanwhile, 137 proteins were identified to be differentially expressed according to the proteomic analysis of EOM samples from 3 TAO patients and 3 control donors, among which VCAN was up-regulated and LCN1 was down-regulated. Moreover, western blotting verified that EOM samples from TAO patients had markedly higher expressions of VCAN and lower expressions of LCN1. GO functional enrichment analysis showed that VCAN was related to proteoglycans and cell recognition, whereas LCN1 was related to retina homeostasis and negative regulation of hydrolase activity. Moreover, differentially expressed proteins were significantly involved in 12 KEGG pathways, such as tight junction, protein digestion and absorption, focal adhesion, ECM-receptor interaction, and cell adhesion molecules. Pappa et al. revealed increased expressions of adhesion molecules in EOM biopsies harvested from TAO patients, whereas minimal or absent expression was observed in control patients (Pappa et al., 1997). In our study, pathway enrichment

analysis showed up-regulated proteins (such as VCAN, MPZ, and PTPRC) were involved in cell adhesion molecules, which is consistent with the previous study. Tight junction was also associated with up-regulated proteins (such as MYH1, MYH4, MYH6, MYH8, MYH3, MYH13 and ACTN3). According to previous investigations about distribution of myosin heavy chain isoforms, which are essential for regulating contraction force and velocity of muscle fibers, normal human EOMs are mainly composed of MYH1, MYH2, and MYH13 (Kjellgren et al., 2003; Park et al., 2012; Schiaffino and Reggiani, 2011). In addition to the up-regulations of MYH1 and MYH13, the other myosin heavy chain isoforms MYH4, MYH6; MYH8, MYH3 were also up-regulated in EOM samples from TAO patients in our study. Moreover, functional analysis for differentially expressed proteins without mRNA transcript alteration showed that MYH3, MYH8, ACTN3, and TNNT1 were related to striated muscle contraction. Thus, it could be speculated that overexpressed MYH1, MYH4, MYH6, MYH8, MYH3, MYH13 and ACTN3 may contribute to abnormal regulations of contraction force and velocity of muscle fibers in TAO. Additionally, ECM-receptor interaction was highly associated with down-regulated proteins (such as COL1A1, COL1A2, and COL2A1), suggesting that ECM-receptor interaction was suppressed in TAO. Meanwhile, COL1A1, COL1A2, and COL2A1 without change in gene levels were related to MET promotes cell motility, collagen formation, MET activates PTK2 signaling, and degradation of the ECM. ECM is composed of fibrillar and interstitial collagens, elastic fibers, and glycoproteins (such as laminin, fibronectin, and proteoglycans) (Hynes, 2009). Aberrant expressions of ECM components derived from tissue-remodelling processes are important for immune cell migration into inflamed tissues, immune cell differentiation, activation, and survival, thereby actively contributing to immune responses (Sorokin, 2010). TAO is an orbital inflammatory disease characterized by OFs activation and proliferation, accumulated deposition of ECM, and adipocyte differentiation and proliferation (Hatton and Rubin, 2002; Naik et al., 2008). Thus, reduced protein expression levels of COL1A1, COL1A2, and COL2A1 may contribute to deposition of ECM in TAO by inhibiting ECM-receptor interaction and degradation of the ECM. It has been documented that glycosaminoglycan accumulates in the retrobulbar

space of patients with TAO (Kahaly et al., 1992). At the same time, we found that VCAN without altered gene expression was involved in ECM proteoglycans and diseases associated with glycosaminoglycan metabolism, suggesting that VCAN is important for TAO by influencing glycosaminoglycan metabolism. However, the gene expression levels of TSHR, IGF-1R, IL-6, and IL-10 were not significantly different in EOM samples between TAO patients and control individuals in our study. It has been reported that the number of TSHR immunostained cells in EOM samples was high in early disease, while decreased with disease duration (Boschi et al., 2005). Wakelkamp et al. have found that the mRNA level of TSHR in orbital fat was largely present only during the active stage but lost in inactive TAO (Wakelkamp et al., 2003). Meanwhile, although elevated levels of IGF-1R have been found in orbital fibroblasts, B and T cells from patients with Graves’ orbitopathy (Smith et al., 2012), the changes in mRNA expression of IGF-1R in EOM samples between TAO patients and control individuals were seldomly reported. According to cytokine profiles in eye muscle tissue, mRNA levels of interferon-γ, TNF-α, IL-1b, and IL-6 were mainly detected in eye muscle tissue, whereas IL-4 and IL-10 mRNA were detected in only one patient out of 14 patients (Hiromatsu et al., 2000). It also has the possibility that because of the inflammation process, especially later stages of the disease, biopsies might have been taken from an area not infiltrated by activated T cells (Pappa et al., 1997). Compared with patients with inactive Graves’ ophthalmopathy, patients with active Graves’ ophthalmopathy had higher mRNA levels of the proinflammatory cytokines including IL-1β, IL-6, IL-8, and IL-10 in the orbital fat/connective tissue (Wakelkamp et al., 2003). In our study, the enrolled TAO patients were in inactive phase with an average clinical course of 36.8 (range, 23~96) months. Thus, it could be possible that no difference was observed in the mRNA expression levels of TSHR, IGF-1R, IL-6, and IL-10, even though these cytokines have been revealed to play important roles in the development of TAO. In conclusion, 952 genes and 137 proteins were identified to be differentially expressed based on transcriptomics and proteomic analysis

of EOM samples from TAO patients and normal controls. Meanwhile, 96 differentially expressed proteins without changes in mRNA expression were uncovered according to the integrative analysis. Differentially expressed 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 ECM (such as COL1A1, COL1A2, COL2A1, VCAN, OGN, and DCN) were identified in EOM samples of TAO. In consistent with the proteomic results, gene microarray analysis identified that the expressions of genes involved in cell adhesion and ECM metabolism were significantly different between EOM samples of TAO patients and controls. The up-regulated expression of VCAN involved in ECM proteoglycans and diseases associated with glycosaminoglycan metabolism, and the down-regulated expression of LCN1 in EOM samples of TAO patients were verified by western blotting. Taken together, this study revealed the significantly altered expression levels of proteins associated with the composition and contraction of muscle fibers, and the proteins and/or genes related to cell adhesion, and the elements and metabolism of ECM, which might suggest the pathogenesis and/or consequences of TAO. Further studies based on this one to elucidate the precise pathological processes of TAO are warranted, which might help to identify potential targets for TAO treatment. Declaration of interests None. Acknowledgements This study was supported by the National Natural Science Foundation of China (81600765 to L.Q.W., 81525006, 81670864 and 81730025 to C.Z.), the Foundation of Shanghai Municipal Commission of Health and Family Planning (201640120 to L.Q.W.), Shanghai Outstanding Academic Leaders (2017BR013 to C.Z.), Excellent Academic Leaders of Shanghai (18XD1401000 to C.Z.), and

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Figure captions Figure 1. Differentially expressed genes in extraocular muscles (EOM) of patients with thyroid-associated ophthalmopathy (TAO) by comparing with control samples from donors. (A) Differentially expressed genes were identified with the thresholds of p value < 0.05 and |log2fold change (FC)| > 1. Red dots represent up-regulated genes, and green ones indicate down-regulated genes. (B) Hierarchical clustering analysis of differentially expressed genes. Higher abundance is colored in pink, the lower ones in blue. Column descriptors indicate the 5 samples per group. C: control samples, T: TAO samples. Figure 2 Principal component analysis (PCA) and hierarchical cluster analysis of differentially expressed proteins associated with TAO. (A) Differentially expressed proteins were identified with the thresholds of p value < 0.05 and |log2FC| > 1. Red dots represent up-regulated proteins, and green ones indicate down-regulated proteins. (B) Scatter plots of the principal component analysis where green dots represent EOM samples from TAO patients and blue squares represent EOM samples from control individuals. (C) Hierarchical clustering analysis of differentially expressed proteins. Higher abundance is colored in red, the lower ones in blue. Column descriptors indicate the 3 samples per group. C: control samples, T: TAO samples. Figure 3. Functional annotations and pathway enrichment for differentially expressed proteins. (A) Gene Ontology (GO) annotation using the Database for Annotation, Visualization and Integrated Discovery (DAVID) with p value < 0.05. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched for differentially expressed proteins. (C) Gene set enrichment analysis (GSEA) for enriched KEGG pathways. Tight junction and cell adhesion molecules were mostly associated with up-regulated proteins. ECM-receptor interaction was highly associated with down-regulated proteins. Figure 4. Constructed protein-protein interaction (PPI) network by search tool for the retrieval of interacting genes (STRING) with minimum required interaction score of 0.4. (A) PPI network for up-regulated differentially expressed proteins. (B) PPI network for

down-regulated differentially expressed proteins. Figure 5. Integrative transcriptomics and proteomic analysis. (A) Protein quantification data and mRNA expression data were annotated to gene symbols. Dots in areas indicated by red arrows were differentially expressed proteins without significantly changed mRNA abundance. (B) Functional annotations for differentially expressed proteins without mRNA transcript alteration. Figure 6. Western blotting analysis of lipocalin 1 and versican expressions in EOM samples from TAO patients and control individuals. C: control samples, T: TAO samples. Supplementary figure 1. Functional annotations for differentially expressed genes. GO annotation using the DAVID with p value < 0.05. Supplementary figure 2. Pathway enrichment for differentially expressed genes. KEGG pathways were enriched for differentially expressed genes using the DAVID with p value < 0.05.

Table 1. The top 15 up-regulated and down-regulated differentially expressed proteins in extraocular muscular samples of thyroid-associated ophthalmopathy patients. Differentially expressed

Proteins

Description

Log2 (fold change)

P value

Up-regulated

MZB1 MYH8 CSRP3 MYH13 IKBKB CD53 NAIF1 RPS4Y1 MYH1 MYH4 SMTNL1 ANK1 MPZ SERPINE2 VCAN PTPRN2 COL8A2 KRT4 COL11A2 MMP10 LCN1 ALDH3A1 KRT13 KRT5 ADH7

Marginal zone B- and B1-cell-specific protein Myosin-8 Cysteine and glycine-rich protein 3 Myosin-13 Inhibitor of nuclear factor kappa-B kinase subunit beta Leukocyte surface antigen CD53 Nuclear apoptosis-inducing factor 40S ribosomal protein S4, Y isoform 1 Myosin-1 Myosin-4 Smoothelin-like protein 1 Ankyrin-1 Myelin protein P0 Glia-derived nexin Versican core protein Receptor-type tyrosine-protein phosphatase N2 Collagen alpha-2(VIII) chain Keratin, type II cytoskeletal 4 COL11A2 Stromelysin-2 Lipocalin-1 Aldehyde dehydrogenase, dimeric NADP-preferring Keratin, type I cytoskeletal 13 Keratin, type II cytoskeletal 5 Alcohol dehydrogenase class 4 mu/sigma chain

2.31 1.90 1.86 1.80 1.80 1.74 1.73 1.72 1.64 1.57 1.56 1.43 1.35 1.34 1.31 -2.83 -2.74 -2.61 -2.54 -2.39 -2.38 -2.29 -2.28 -2.24 -2.20

2.90E-07 1.24E-06 1.49E-05 5.34E-07 5.22E-08 3.20E-05 1.36E-04 1.72E-05 6.90E-07 2.15E-07 4.56E-06 2.61E-05 2.80E-09 3.78E-05 8.75E-07 1.43E-02 5.33E-08 3.14E-08 1.77E-07 4.92E-06 1.12E-06 2.47E-08 1.08E-07 8.97E-08 1.46E-07

Down-regulated

PAWR ANGPTL7 SORBS2 COL2A1 SFN

PRKC apoptosis WT1 regulator protein Angiopoietin-related protein 7 Sorbin and SH3 domain-containing protein 2 Collagen alpha-1(II) chain 14-3-3 protein sigma

-2.17 -2.16 -2.10 -2.07 -2.06

2.06E-06 2.18E-07 1.08E-06 3.71E-06 2.99E-07

Table 2. Gene Ontology (GO) annotations for 137 differentially expressed proteins. GO ID

Description

Log10P

Z-score

Count

M5884

NABA CORE MATRISOME

-13.78

13.45

18

GO:0097435

Supramolecular fiber organization

-12.58

10.77

24

GO:0070268

Cornification

-11.85

14.51

12

GO:0030049 GO:0045110 M5882

Muscle filament sliding Intermediate filament bundle assembly NABA PROTEOGLYCANS

-8.74 -7.50 -7.43

14.64 20.16 13.22

7 4 6

GO:0006959

Humoral immune response

-6.13

7.21

12

GO:0042060

Wound healing

-5.55

6.29

14

GO:0001895 GO:0008037

Retina homeostasis Cell recognition

-5.52 -5.48

8.90 7.20

6 9

GO:0051346

Negative regulation of hydrolase activity

-4.97

5.97

12

GO:0051209

Release of sequestered calcium ion into cytosol

-4.17

6.44

6

GO:0008285

Negative regulation of cell proliferation

-4.02

4.81

14

GO:0030155

Regulation of cell adhesion

-3.93

4.79

13

GO:0007044 GO:0061436

Cell-substrate junction assembly Establishment of skin barrier

-3.70 -3.69

6.15 8.47

5 3

Genes COL1A1, COL1A2, COL2A1, COL8A2, COL11A2, COL16A1, VCAN, DCN, DPT, FMOD, OGN, PRELP, SRPX, POSTN, KERA, SMOC1, COL6A6, CILP2 COL1A1, COL1A2, COL2A1, COL11A2, DPT, FMOD, KRT8, KRT9, KRT14, KRT19, MYH3, MYH6, MYOC, NEFH, NEFL, PAWR, TNNT1, TPM1, CSRP3, SORBS2, MYOM2, CORO1A, EPPK1, SCIN CSTA, KRT1, KRT4, KRT5, KRT7, KRT8, KRT9, KRT13, KRT14, KRT15, KRT19, PPL ACTN3, MYH3, MYH4, MYH6, MYH8, TNNT1, TPM1 KRT14, NEFH, NEFL, EPPK1 VCAN, DCN, FMOD, OGN, PRELP, KERA IGHD, KRT1, LTF, LYZ, PTPRC, SLPI, IGHV6-1, IGHV3-43, IGLV3-25, IGLV3-19, IGLC7, IGKV2-40 COL1A1, COL1A2, DCN, GAP43, GPR4, HBG2, KRT1, SERPINE2, PPL, TPM1, PROCR, POSTN, EPPK1, LACRT CST4, KRT1, LCN1, LTF, LYZ, PIP BSG, VCAN, GAP43, IGHD, CORO1A, IGHV6-1, IGHV3-43, IGLC7, NTM CST4, CSTA, SFN, IKBKB, LCN1, LTF, SERPINB5, SERPINE2, PLN, PTN, PTPRN2, SLPI PLN, PTPRC, CORO1A, PLCH1, LACRT, DHRS7C CAV2, CNN1, DPT, SFN, KRT4, OGN, PAWR, SERPINE2, PTN, TPM1, SRPX, TNMD, EPPK1, SCIN COL1A1, COL16A1, DPP4, IL1RN, MYOC, PAWR, SERPINE2, PTN, PTPRC, TPM1, POSTN, CORO1A, TENM3 ACTN3, COL16A1, KRT5, KRT14, MYOC SFN, KRT1, HRNR

R-HSA-6798695 GO:0071363 GO:0016311 GO:0021762

Neutrophil degranulation Cellular response to growth factor stimulus Dephosphorylation Substantia nigra development

-3.45

4.55

10

-3.24

4.16

12

-2.88 -2.76

3.99 5.67

9 3

CD53, HLA-B, KRT1, LTF, LYZ, PTPRC, PTPRN2, SLPI, PADI2, HRNR CAV2, COL1A1, COL1A2, COL2A1, DCN, FMOD, MT1G, MYH6, PTN, POSTN, CORO1A, TNMD EPHX2, IKBKB, MYH3, MYH6, MYH8, PTN, PTPRC, PTPRN2, CILP2 PLP1, S100A1, PADI2

Table 3. Significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for 137 differentially expressed proteins. Pathway ID

Pathway Name

Genes

Count

hsa04530 hsa04974 hsa04510 hsa04512 hsa05204 hsa00350 hsa05014 hsa04514 hsa00010 hsa04151 hsa04115 hsa04260

Tight junction Protein digestion and absorption Focal adhesion ECM-receptor interaction Chemical carcinogenesis Tyrosine metabolism Amyotrophic lateral sclerosis Cell adhesion molecules Glycolysis / Gluconeogenesis PI3K-Akt signaling pathway p53 signaling pathway Cardiac muscle contraction

MYH1, MYH4, MYH6; MYH8, MYH3; MYH13, ACTN3, MYL10 COL1A1, COL1A2, COL6A6, DPP4, COL2A1 COL1A1, COL1A2, COL6A6, ACTN3, MYL10, COL2A1, CAV2 COL1A1, COL1A2, COL6A6, COL2A1 ALDH3A1, GSTM5, ADH7 ALDH3A1, ADH7 NEFL, NEFH VCAN, MPZ, PTPRC ALDH3A1, ADH7 COL1A1, COL1A2, COL6A6, COL2A1, IKBKB SFN, SERPINB5 MYH6, COX7C

8 5 7 4 3 2 2 3 2 5 2 2

Supplementary table 1. Demographic results and clinical profiles of TAO patients and controls. TAO

Control

Number

11

11

Gender (male/female)

7/4

8/3

Age (median, range), years

49.8 ± 5.8 (49, 40~59)

43.9 ± 8.9 (45, 23~ 58)

Duration of TAO (median, range), months

36.8 ± 21.1 (30, 23~96)

N/A

Clinical activity score (median, range）

0.6 ± 0.8 (0, 0~2)

N/A

T3 (median, range), nmol/L

1.71 ± 0.41 (1.72, 1.26~2.74)

N/A

T4 (median, range), nmol/L

92.13 ± 16.10 (86.09, 76.19~123.8)

N/A

FT3 (median, range), pmol/L

4.76 ± 0.59 (4.79, 3.56~5.67)

N/A

FT4 (median, range), pmol/L

16.50 ± 2.40 (16.61, 13.27~21.66)

N/A

TSH (median, range), mIU/L

2.09 ± 1.19 (1.96, 0.48~3.98)

N/A

Supplementary table 2. GO annotations for 952 differentially expressed genes. GO ID

Description

Count

P Value

GO:003059 3

neutrophil chemotaxis

13

4.41E-05

GO:000715 5

cell adhesion

42

4.57E-05

UDP-N-acetylglucosamine biosynthetic process

5

1.16E-03

RENBP, UAP1, GNPDA1, GFPT2, UAP1L1

defense response to fungus

7

1.24E-03

S100A8, S100A9, DEFA4, DEFA1, HTN3, IL17RA, S100A12

chemotaxis

15

1.55E-03

immune response

34

2.30E-03

regulation of muscle contraction

5

3.15E-03

TNNT2, ANXA6, MYL5, TNNC1, PPP1R12B

icosanoid metabolic process

4

4.66E-03

CYP2J2, PTGIS, CYP4F22, CYP4F2

10

6.41E-03

CCL23, CCL14, S100A8, ZP3, IL18, S100A9, CCL8, CCL5, CCL4, S100A12

5

6.71E-03

WFDC10B, CST3, CSTA, PI16, ECM1

7

7.53E-03

EPHA5, ADCY2, ADCY7, ADCY6, NPR3, DEFB1, PCLO

GO:000604 8 GO:005083 2 GO:000693 5 GO:000695 5 GO:000693 7 GO:000669 0 GO:005072 9 GO:001046 6 GO:001993

positive regulation of inflammatory response negative regulation of peptidase activity cAMP-mediated signaling

Genes CCL23, CCL14, C5AR1, S100A8, EDN2, CXCL3, IFNG, S100A9, CCL8, TREM1, CCL5, CCL4, S100A12 ATP1B2, TNC, ITGA11, FER, CCL4, APLP1, CD96, COL6A6, SORBS2, TGFBI, ACAN, CNTNAP3, COL12A1, ROBO2, GP1BA, GPNMB, THBS2, CEACAM1, THBS3, DSCAM, COL4A3, ADGRE1, ADGRE2, SVEP1, CNTN6, ICAM3, PTPRT, CERCAM, CTNNA1, TPBG, CTNNA2, OMD, CD36, CD34, LSAMP, CDON, CNTN4, SEMA4D, ADAM12, CHL1, NTM, ITGA2B

RNASE2, C5AR1, CMTM2, ENPP2, CCL8, FER, ACKR4, CCL5, CXCL11, PROK2, DOCK2, CCL23, DEFA1, XCR1, DEFB1 TNFRSF6B, ENPP2, CXCL3, IL18, CCL8, CCL5, CXCL11, CCL4, CD96, CCL23, IFNG, OTUD7B, SEMA3C, DEFB1, FYB, IL18R1, CRIP1, CR2, C5AR1, IL7, GZMA, CTLA4, ACKR4, HLA-DQA1, TNFRSF10C, RGS1, CD36, CCL14, CST7, CD40LG, SLPI, DEFA1, SEMA4D, CNIH1

3 GO:000718 9 GO:000257 6 GO:003249 6 GO:000720 4 GO:000254 8 GO:000715 6 GO:007009 8 GO:006004 5 GO:000695 4

adenylate cyclase-activating G-protein coupled receptor signaling pathway

8

7.84E-03

platelet degranulation

12

8.13E-03

response to lipopolysaccharide

16

9.30E-03

positive regulation of cytosolic calcium ion concentration

14

9.35E-03

monocyte chemotaxis

7

1.23E-02

CALCA, CCL23, CCL14, CCL8, CCL5, CCL4, S100A12

15

1.52E-02

PLXNB3, PCDH12, PCDHGA6, PTPRT, PCDHB13, PCDH8, PCDHGA4, PCDHGA3, CDH9, FAT3, TENM3, ROBO2, IGSF9, CEACAM1, DSCAM

9

1.66E-02

CCL23, CCL14, CXCL3, CCL8, ACKR4, CCL5, CXCL11, XCR1, CCL4

5

1.72E-02

FGF9, TBX5, ZFPM2, RBPJ, NRG1

1.79E-02

TNFRSF6B, ABCF1, S100A8, CXCL3, IL18, CRHBP, S100A9, CCL8, CXCL11, CCL5, CCL4, CALCA, CCL23, XCR1, ADGRE2, C5AR1, NFRKB, CD5L, CELA1, PTGFR, ECM1, S100A12, PROK2, TNFRSF10C, CCL14, CD40LG, AOX1, CLEC7A

homophilic cell adhesion via plasma membrane adhesion molecules chemokine-mediated signaling pathway positive regulation of cardiac muscle cell proliferation

inflammatory response

28

CALCA, PTHLH, ADCY2, ADCY7, DRD5, ADCY6, NPR3, PTGFR CD36, BRPF3, CLEC3B, CLU, HABP4, SERPING1, CFD, EGF, ECM1, TIMP3, SRGN, ITGA2B TNFRSF6B, C5AR1, S100A8, CXCL3, CNP, FER, CXCL11, PTGFR, JUNB, TRIB1, CD96, TNFRSF10C, PENK, JUN, SLPI, ABCC8 C5AR1, SWAP70, EDN2, GPR6, CD52, LPAR2, PTGFR, ITPR3, CALCA, PROK2, PLCE1, CD55, CD36, XCR1

Supplementary table 3. The enriched KEGG pathways for 952 differentially expressed genes. Pathway ID

Pathway Name

Count

P Value

hsa04970

Salivary secretion

12

7.14E-04

hsa04512

ECM-receptor interaction

11

2.80E-03

hsa00520

Amino sugar and nucleotide sugar metabolism

8

3.14E-03

hsa04060

Cytokine-cytokine receptor interaction

20

4.99E-03

hsa04610 hsa00590 hsa05144 hsa04911 hsa00980 hsa00512

Complement and coagulation cascades Arachidonic acid metabolism Malaria Insulin secretion Metabolism of xenobiotics by cytochrome P450 Mucin type O-Glycan biosynthesis

9 8 7 9 8 5

6.88E-03 1.18E-02 1.43E-02 2.26E-02 3.12E-02 3.64E-02

hsa04062

Chemokine signaling pathway

14

4.16E-02

hsa04921

Oxytocin signaling pathway

12

4.36E-02

hsa01100

Metabolic pathways

62

4.94E-02

Genes ADCY2, ADCY7, PRH2, ATP1B2, STATH, ADCY6, CST3, GUCY1A2, SLC4A2, VAMP2, ITPR3, HTN3 COL4A3, CD36, COL6A6, TNC, ITGA11, SV2B, GP1BA, SV2A, THBS2, THBS3, ITGA2B RENBP, UAP1, GNPDA1, MPI, GFPT2, UGDH, UAP1L1, NANP TNFRSF6B, IL18R1, IL7, LEPR, CXCL3, IL18, CCL8, CXCL11, CCL5, CCL4, IL17RA, TNFRSF10C, CCL23, CCL14, IL20RB, CD40LG, IFNG, BMP7, XCR1, THPO PLAT, CD55, F10, CR2, C5AR1, CFB, F2, SERPING1, CFD GPX2, CYP2J2, PTGIS, CYP2C9, PLA2G2C, GGT1, CBR3, CYP4F2 CD36, CD40LG, IL18, IFNG, THBS2, THBS3, SDC2 ADCY2, ADCY7, ATP1B2, ADCY6, KCNN2, VAMP2, ITPR3, ABCC8, PCLO CYP3A5, AKR1C2, CYP2C9, HSD11B1, ADH1C, ADH1A, CBR3, AKR1C1 GALNTL5, GALNT15, GALNT8, GALNT12, GALNT13 ADCY2, ADCY7, CXCL3, ADCY6, CCL8, CXCL11, CCL5, CCL4, DOCK2, CCL23, CCL14, GRK5, SHC3, XCR1 ADCY2, ADCY7, CACNG8, JUN, PPP1R12B, PRKAB2, ADCY6, GUCY1A2, EEF2K, ELK1, NFATC2, ITPR3 CYP3A5, GNPDA1, CYP2J2, CNDP2, ADH1C, ADH1A, PPOX, GGT1, MTHFD1L, PTGIS, GALNTL5, ST3GAL6, NANP, NMNAT3, ACSM2A, PFKP, FBP2, CBR3, TAT, COQ5, PLCE1, UMPS, DHRS4, HYKK, SDS, RRM1, HSD11B1, KDSR, PLA2G2C, MVK, UROC1, AMY1C, GALNT8, EXT1, BCAT1, HSD17B2, FUT7, UGDH, ASL, POLR2A, HPSE2, CERS2, GALNT15, HAAO, MTMR8, BDH2, GALNT12, GALNT13, EBP, UAP1, CYP2C9, AK1, NTPCR, UAP1L1, DBH, MPI, SDHC, GFPT2, AOX1, CYP4F2, LIPC, ABO

Volcano Plot ●

FC=2

6

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● ● ● ● ● ● ● ●●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●●● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

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P value=0.05

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1

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0

−log10(P value)

● ●

−5

−4

−3

−2

−1

0 log2(FC)

1

2

3

4

5

Samples Group Samples 2 C1 C2 1 C3 C4 0 C5 T1 −1 T2 T3 T4 −2 T5 Group TAO Normal

T5

T3

T4

T2

T1

C5

C2

C4

C3

C1

Volcano Plot ●

8

FC=1/2 ● ● ● ● ● ●

6

●

2

4

●

0 −5

−4

−3

● ● ●

● ● ●

●

●

●

●

● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ●● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ●● ●● ● ● ●●● ● ●●● ● ●● ● ●● ●● ● ● ● ● ●●● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ●● ● ●●●● ● ● ● ● ● ● ● ● ● ● ●● ●● ●● ● ● ● ●●●● ● ● ● ● ●●●● ● ●●● ● ● ●● ●● ● ●●● ● ●●●● ● ● ●● ●●● ● ● ●●● ● ● ● ● ●● ● ●● ● ●● ● ● ●● ● ● ●● ● ● ●● ● ● ● ●● ● ●● ● ●● ●●● ●●● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ●●● ●● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ●● ●● ● ● ● ●●● ● ●● ● ● ● ● ●● ●●●●●● ● ● ●● ● ● ● ● ●● ● ● ● ●● ●● ●● ● ● ● ●●● ● ● ● ● ● ●● ● ● ● ●● ● ● ●● ● ● ●● ● ●● ● ● ● ● ● ● ●●● ● ●● ●● ●● ● ●● ● ● ● ●● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ●● ● ● ● ● ●● ● ● ● ●●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●●● ●● ● ●● ● ●● ●● ● ● ● ● ● ●●●●● ● ● ● ● ● ● ● ● ● ●●● ●●● ● ● ●● ● ● ●● ● ●● ● ●●●● ● ●●● ●● ● ●● ●● ●●●● ● ●●● ●● ● ● ●●● ● ● ● ●● ●● ● ●● ●● ●● ●●● ● ● ● ●●●● ● ● ● ● ●● ●● ● ● ● ●● ● ● ●● ● ●●● ● ●● ● ●● ● ● ● ● ● ● ● ●● ● ●●●● ● ●● ● ● ● ●● ●● ● ● ● ●● ●●● ● ●● ● ●● ● ● ● ●●● ● ● ● ●●● ● ●●●●●●●● ●●● ● ● ● ● ●●● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●●● ● ● ● ● ●● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●●● ● ● ● ● ● ● ●● ●● ● ●●●● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ●● ● ●● ●● ● ● ● ● ● ●●● ● ● ● ●● ●●● ●● ●● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ●● ●●●● ●● ● ●● ● ● ● ●● ● ●● ● ● ●● ● ●●● ●● ●● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ●● ●●● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●●●● ● ●● ●●● ●● ● ●● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ●● ●●●●● ● ●● ● ● ●● ●●● ● ●● ●● ● ● ● ● ● ● ● ●● ● ●●●● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ●● ● ● ● ●● ●● ● ● ●●●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ●● ● ●● ● ●●● ●● ● ● ● ● ● ● ● ●●● ● ● ●● ● ●● ● ●●● ● ● ●● ● ● ● ● ●● ● ● ● ●●● ●● ●● ● ●● ● ● ● ● ● ● ● ●● ●● ●● ●● ●● ●●●● ● ● ● ●●●●● ● ● ●●● ● ● ●●● ●● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ●● ● ● ● ●●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ●● ●●● ●● ●● ● ●● ● ● ●● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ●● ● ● ●●●● ● ● ●● ●● ● ●● ●● ● ●● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ●● ● ● ●● ● ●●● ● ● ● ● ● ●●● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ●●● ● ● ● ● ● ●● ● ● ● ● ● ● ●●●● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ●●●● ● ● ● ●● ● ● ● ● ● ●●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ●● ● ●●●● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ●● ● ● ● ●● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ●●● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ●● ● ● ●● ● ● ●● ●●●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●●●●● ● ● ● ● ●● ● ● ● ● ●●●●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●●●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ●●●● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ●● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ●●● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●●● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●

●

●

●

FC=2

●

● ● ● ● ● ● ●

● ●

−log10(p−value)

●

● ●

●

●

−2

−1

0 log2(FC)

1

2

●

p−value=0.05

3

4

5

Samples Group

1.5

TENM3_Q9P273 S100A1_Q5T7Y6 PLCH1_Q4KWH8 TUBB3_Q13509 TNNT1_P13805 NDUFB1_O75438 CD53_P19397 POSTN_B1ALD9 ANK1_H0YBS0 SSR2_P43308 RPS4Y1_P22090 DHRS7C_A6NNS2 PTPRC_P08575 PADI2_Q9Y2J8 SMTNL1_E9PPJ3 RPL32_F8W727 SEC11C_Q9BY50 FKBP11_Q9NYL4 MYH4_Q9Y623 MYH1_P12882 MYOM2_P54296 MZB1_Q8WU39 MPZ_P25189 IKBKB_O14920 HIST1H1B_P16401 NNMT_P40261 MYH6_P13533 ACTN3_Q08043 MYH3_P11055 MYH8_P13535 NEFL_P07196 MYH13_Q9UKX3 VCAN_P13611 SSBP1_Q04837 CORO1A_P31146 CSRP3_P50461 HLA−B_Q29836 SERPINE2_P07093 COX7C_P15954 NEFH_P12036 NAIF1_Q69YI7 IGHD_A0A0A0MS09 PTPRN2_Q92932 IRGQ_Q8WZA9 GPR4_P46093 ZNF512_Q96ME7 CLEC11A_Q9Y240 IGKV1−6_A0A0C4DH72 EPPK1_P58107 IGKV2D−24_A0A075B6R9 ZNF185_O15231 SMOC1_Q9H4F8 ENPP6_Q6UWR7 PTN_P21246 MYL10_Q9BUA6 SCGB1D1_O95968 PAWR_Q96IZ0 HBG2_P69892 LTF_P02788 KRT1_P04264 IGHV3−43_A0A0B4J1X8 IGKV2−40_A0A087WW87 MRPS18A_Q5QPA5 MAMDC2_Q7Z304 SERPINB5_P36952 TPM1_H0YNC7 SCIN_Q9Y6U3 KRT15_P19012 EPHX2_P34913 FAM180B_Q6P0A1 IGHV3OR15−7_A0A075B7D8 SFN_P31947 MYOC_Q99972 LACRT_Q9GZZ8 KRT8_P05787 CNN1_P51911 IGLC7_A0M8Q6 ALDH3A1_P30838 COL6A6_A6NMZ7 KRT4_P19013 KRT13_P13646 KRT5_P13647 CAV2_P51636 SCGB2A1_O75556 ADH7_P40394 OGN_P20774 SLPI_P03973 FMOD_Q06828 CILP2_K7EPJ4 PRELP_P51888 KRT7_P08729 COL16A1_Q07092 ANGPTL7_O43827 GAP43_P17677 COL11A2_A0A0C4DFS1 LCN1_P31025 MT1G_P13640 KRT14_P02533 COL2A1_P02458 KRT19_P08727 COL1A1_P02452 DCN_P07585 COL1A2_P08123 PPL_O60437 IGLV3−25_P01717 SORBS2_H7BXR3 GSTM5_P46439 CPXM2_Q8N436 HRNR_Q86YZ3 DPP4_P27487 LYZ_P61626 KERA_O60938 COL8A2_P25067 SORBS2_O94875 PIP_P12273 KRT9_P35527 ADIRF_Q15847 DPT_Q07507 CST4_P01036 SRPX_P78539 MUC5AC_P98088 SRD5A3_Q9H8P0 IGLV3−19_P01714 IL1RN_P18510 NTM_Q9P121 PLP1_P60201 PLN_P26678 CSTA_P01040 FCGBP_Q9Y6R7 MMP10_P09238 PVALB_P20472 BSG_P35613 WNT2B_Q93097 TNMD_Q9H2S6 IGHV6−1_A0A0B4J1U7 TNFSF12−TNFSF13_A0A0A6YY99 PROCR_Q9UNN8

1 0.5 0 −0.5

Samples C1 C2 C3 T1 T2 T3

Group Case Control −1.5 −1

C3

C2

C1

T3

T1

T2

COL1A2

COL2A1 COL16A1

Degradation of the extracellular matrix

COL1A1

Collagen chain trimerization

DCN

Non-integrin membrane-ECM interactions MET promotes cell motility

Integrin cell surface interactions

BSG

MET activates PTK2 signaling

Collagen degradation OGN

Assembly of collagen fibrils and other Collagen multimeric formation structures MYH3

TNNT1

Collagen VCAN ECM ACTN3 Diseases Striated Muscle associated with biosynthesis proteoglycans Contraction and modifying glycosaminoglycan MYH8 enzymes metabolism

Highlights 1. Proteomic analysis of EOM samples from TAO patients and control individuals revealed 137 differentially expressed proteins, which were mainly related to cellular components of EOM, muscle contraction, cell adhesion and ECM metabolism. 2. Gene microarray analysis of EOM samples from TAO patients and controls identified 952 differentially expressed genes significantly involved in cell adhesion and ECM metabolism. 3. Western blotting verified the up-regulated expression of versican and the down-regulated expression of lipocalin 1 in EOM samples from TAO patients.