Our study indicated that a total of 366 circRNAs were differentially expressed in plasma of GD compared with that of healthy control subjects. Among them, there were 195 circRNAs up-regulated and 171 down-regulated, suggesting that circRNAs might play a crucial regulatory role in pathogenesis of GD.
In the microarray analysis of all different expression of circRNAs, we found that exonic circRNAs accounted for the largest proportion (84.3%) which might be related to their function. The exonic circRNA is a loop containing a 3'-5' phosphodiester bond which was described as a covalent link between the 3' end of an exon and the 5' end of either the same exon or the upstream exon. The specific circle structure protects circRNAs from 'exon skipping' and leads them to become more inclined to regulate the linear coding RNAs from which they are derived [3].
The chromosomal distribution analysis of circRNAs in this study showed that the different expression of circRNAs were mainly localized on chr17, chr2 and chr3. Some studies have indicated that X chromosome disorder was associated with female predominance in adult autoimmune thyroid disease (ATD) [11, 12], however a similar result was not reflected in this chromosomal distribution analysis. This difference may be due to the microarray analysis of small samples to lead to the poor reproducibility of the experimental results. It is therefore necessary to perform a high-quality and large-scale study to further explore this point. Ecsedi et al. showed that chr17 imbalance may lead to cell proliferation activation, supporting our findings [13]. We screened three significantly different expressions of circRNAs that included 2 up-regulated circRNAs (hsa_circ_0090364, hsa_circ_0001228) and 1 down-regulated circRNA (hsa_circ_0008339) for RT-qPCR validation. The results showed that hsa_circ_0090364 was consistent in microarray analysis and RT-qPCR, which indicated that the microarray analysis was highly reliable. In summary, circRNAs may have regulatory functions in the epigenetic regulation mechanism of GD, which deserves further attention.
Hsa_circ_0090364 is a high-expression circular exonic RNA in GD, which is mainly generated by the selective splicing of its parental gene Jade3 exon. Therefore, the higher levels of JADE3 in patients implies the potentially elevated expression of hsa_circ_0090364. According to Circbase’s annotation, JADE3 was involved in inducing histone acetylation during transcription. Although the relationship between histone acetylation and GD has not been discussed, a study has revealed that the high level of histone H3 acetylation (H3ac) increased the level of IL-6 in Rheumatoid Arthritis Synovial Fibroblast (RASFs) to promote rheumatoid arthritis (RA) [14]. In addition, some studies have identified that the high levels of IL-6 were correlated with increased risk for GD [15, 16]. The findings above suggest that JADE3 plays an important role in the pathogenesis of AITD. Until now, the nature of the specific mechanisms between JADE3 and hsa_circ_0090364 remain unclear. To further investigate the upstream-regulatory mechanism of circRNAs, this study indirectly obtained the transcription factors of hsa_circ_0090364 by predicting the transcription factors of JADE3. As the predicted results show, a total of 3608 transcript factors were predicted. We focused on the 24 transcript factors which have been confirmed by three or more experiments. Functional annotations of these transcript factors suggested that they could promote cell proliferation, differentiation and immune inflammation. Furthermore, the pathogenesis of GD was also related to these biological processes [2]. Given the above studies, we speculated that there was a regulatory relationship among transcript factors, parental gene and circRNAs in the development of GD, with further studies required to illustrate this in detail.
In the ceRNA network of hsa_circ_0090364, we found that hsa_circ_0090364 interacted with multiple miRNAs in the miR-378 family (miR-378h, miR-378f, miR-378b, miR-378c, miR-378a-3p). The 2D structure of microRNA and circRNA (Fig. 6) showed that the binding site of miR-378a-3p, miR-378c and miR-378f on hsa_circ_0090364 had the same microRNA seed sequence CUGGAC, which was able to match perfectly with hsa_circ_0090364. The microRNA seed sequence is defined as nucleotides 2–7 of the 5' region on the microRNA, and is believed to be the most conserved section of the microRNAs, which is of great importance for miRNA recognition [17]. The same microRNA seed sequence further confirmed that hsa_circ_0090364 and each member of miR-378 family had the same binding ability. Therefore, we presume that hsa_circ_0090364 acts as an ‘endogenous sponge’ for many microRNAs from the miR-378 family by using the MRE as ‘RNA language’.
The miR-378 family has not been reported in studies of GD patients, but was reported by Shi Haizhong et al [18]. It is suggested that the low expression of the miR-378 family enhanced the levels of its downstream molecule IL-33 and played a role in the proinflammatory response of the autoimmune disease ulcerative colitis (UC). At the same time, the investigators screened for miR-378a-3p to verify it. In the present study, hsa_circ_0090364 was detected to be significantly up-regulated in GD, and it was established that pathogenesis of GD was the process involved in the autoimmune inflammatory response leading to thyroid cell proliferation resulting in thyroid hormone secretion. Therefore, we hypothesized that hsa_circ_0090364 may act as an endogenous sponge of miR-378h, miR-378f, miR-378b, miR-378c and miR-378a-3p. When hsa_circ_0090364 was up-regulated in GD, miR-378h, miR-378f, miR-378b, miR-378c and miR-378a-3p were repressed at the transcriptional level, thereby regulating the target genes and prompting the occurrence and development of GD. However, this hypothesis needs to be verified by future study.
The GO functional analysis of the ceRNA network showed that multiple biological processes of GO enrichment in the network were related to multiple metabolic processes, such as activation of the MAPK pathway, phosphatidylinositol-mediated signal transduction and IGF-1-IGF1R complex synthesis. The MAPK pathway plays a key role in inducing protein synthesis and cell differentiation. Thus, stimulating antibodies influences thyroid cell activation and proliferation by modulating MAPK signalling cascades in GD. PI3Ks are downstream signal molecules of phosphatidylinositol. The PI3K pathway has the function of regulation of thyroid cell proliferation and hormone synthesis [19]. The IGF-1-IGF1R complex is the core component of the development of thyroid-associated ophthalmopathy. Thyroid stimulating immunoglobulins activate the IGF-1-IGF1R complex through TSHR-IGF1R-cross-talk and increase the level of glycosaminoglycan synthesis and inflammatory molecules. Moreover, immuno-globulins against the IGF1R can activate signals in orbital fibroblasts, leading to the production of hyaluronic acid and cytokines [20]. In the KEGG pathways analysis of circRNA-miRNA-mRNA network, activating the T cell receptor pathway was one of multiple enriched pathways. In GD, excessive thyroid hormones are secreted by thyroid cells due to being stimulated by thyroid stimulating hormone receptor (TSHR) autoantibodies, which are produced by plasmacyte and local B cells controlled by activated T cells. In this process, activating the T cell receptor pathway is crucial [21, 22].
It must be considered that limitations still exist. Firstly, with a greater number of study subjects patient samples used for detection may be increased, and the range of differential expression of circRNAs in GD patients may be narrowed. Secondly, this study confined its analysis to the different mapping of circRNAs and explored their potency to act as internal competitive endogenous RNAs (ceRNAs), while the functional analysis of circRNAs was not comprehensive. In future studies, more functional validation shall be required.