A total of 626 mRNAs were significantly differentially expressed in the 2 subtype tissues, with 488 mRNAs upregulated and 138 mRNAs downregulated according to RNA-seq analysis. Combined with bioinformatic prediction, 262 mRNAs were identified. To further determine the role of those target genes contained in the ceRNA network, we also performed GO and KEGG analyses, and we obtained 78 BP terms, 37 CC terms, 18 MF terms, and 16 important signaling pathways, many of which were consistent with the current knowledge on TAO. For example, extracellular matrix organization (GO: 0030198) was included as a BP term. Autoantibodies can bind to ECM proteins in vivo, playing an important role in orbital tissue inflammation.
38 Through KEGG analysis, we found that the PI3K-Akt signaling pathway, Th17-cell differentiation, and the relaxin signaling pathway were involved. OFs express TSHR, a G protein-coupled cell surface protein.
39 The activation of TSHR signaling can lead to increased expression of genes involved in inflammation.
40 TSH could induce IL-1RA in OFs by activating the PI3K/AKT pathway.
41 Th17 cells, a subset of helper T (Th) cells, are characterized by the production of cytokines, such as IL-17A and IL-17F. IL-17A has been confirmed to induce OFs to secrete proinflammatory cytokines and promote excessive ECM synthesis and deposition in TAO, dependent on MAPK activation.
42 Relaxin has a molecular weight of 6 kD and is a kind of endogenous hormone synthesized and secreted by several tissues in the human body, including reproductive organs and nonreproductive organs. Relaxin 2 binds to its receptor, RXFP1, via specific pathways, such as the Notch signaling pathway and Wnt signaling pathway, to inhibit fibrosis pathological processes.
43–45 Therefore, these signaling pathways may be critical in the pathogenesis and classification of TAO. KEGG analysis showed that COL1A1 and MMP2, the regulatory genes of hsa_circ_0007006, participated in the relaxin signaling pathway. Furthermore, after PPI network construction and identification of the hub genes with Cytoscape, COL1A1 and MMP2 were found to be among the top 10 hub genes. The mRNA expression levels of COL1A1 and MMP2 were verified in our study. Fibrosis is considered to be the terminal stage of TAO, followed by the inflammation of Muller’s muscle, extraocular muscles, and orbital connective and adipose tissues.
1,46 Excessive deposition of ECM was considered to be associated with this progression. According to the manifestations and characteristics of type I and type II TAO, the apparent distinction between them may involve fibrosis. COL1A1 is a marker of fibrosis.
47 Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) participate in maintaining the balance of the ECM. To better investigate the relationship between hsa_circ_0007006 with COL1A1 and MMP2, a lentivirus was used to overexpress hsa_circ_0007006. We discovered that COL1A1 and MMP2 expression level was reduced compared to the control group. In addition, by performing WB experiments, we found that compared to type I, the relaxin signaling pathway was more activated in type II. In summary, hsa_circ_0007006 may regulate major genes, such as MMP2 and COL1A1, through the relaxin signaling pathway to affect TAO typing. Hence, hsa_circ_0007006 could represent a meaningful diagnostic and prognostic biomarker for TAO subtyping. Additionally, hsa_circ_0007006 has a negative correlation with COL1A1 and MMP2. These findings may present a new breakthrough to treat fibrosis.