Differential Circular RNA Expression Profiling of Orbital Connective Tissue From Patients With Type I and Type II Thyroid-Associated Ophthalmopathy

PURPOSE: The purpose of this study was to investigate the molecular mechanism underlying thyroid-associated ophthalmopathy (TAO) clinical subtypes, to do so, we performed transcriptomic analysis to reveal the expression profile of circular RNAs (circRNAs) in TAO subtypes. METHODS: High-throughput RNA-sequencing was performed in six pairs of type I and type II orbital connective tissue samples from patients with TAO. The expression levels of circRNAs and mRNAs in type I and type II samples were measured by quantitative real-time polymerase chain reaction (qRT-PCR) in another three pairs of type I and type II TAO connective tissue samples. We used bioinformatics predictions to construct a circRNA-microRNA (miRNA)-mRNA network. A protein-protein interaction (PPI) network was constructed based on differential mRNA expression, and the hub genes were determined by the Cytoscape software plugin. Functional and pathway enrichment analyses were performed to elucidate circRNA function. Lentiviral-mediated overexpression of hsa_circ_0007006 and the relationship between hsa_circ_0007006 with COL1A1 and MMP2 were evaluated by Western blotting (WB). Moreover, the differential pathways were assessed by WB. RESULTS: RNA sequencing results predicted a total of 7489 circRNAs and 15,803 mRNAs, with 94 upregulated and 76 downregulated circRNAs and 488 upregulated and 138 downregulated mRNAs. The qRT-PCR analysis of seven dysregulated circRNAs and two major mRNAs validated the RNA-sequencing data. The competing endogenous RNA (ceRNA) network included 7 circRNAs, 23 miRNAs, and 262 mRNAs. Functional analysis revealed several important pathways. Overexpression of hsa_circ_0007006 led to decreased expression levels of COL1A1 and MMP2. Activation of the relaxin signaling pathway differed between the two subtypes. CONCLUSION: We showed that circRNAs are differentially expressed between type I and type II TAO. We speculate that the hsa_circ_0007006-COL1A1 and MMP2-relaxin signaling pathways are important regulatory axes in the pathogenesis of this disease type and could be considered promising diagnostic and therapeutic targets in the future.