Identification of Circular RNAs Circ_0005008 and Circ_0005198 in Plasma as Novel Biomarkers for New-Onset Rheumatoid Arthritis

FIGURE 1. Cluster analysis of the comparison of 10 circRNAs that showed differential expression in 5 new-onset patients with RA compared with their expression levels in 5 HCs. Each row represents an different gene; each column represents an individual sample. “Red” marks high relative expression and “green” marks low relative expression (readers are referred to the web version of this article to see the interpretation of the colors in this figure).

FIGURE 2. qRT-PCR determination of the relative circRNA expression levels in plasma from 40 new-onset patients with RA, 40 healthy controls, and 25 new-onset patients with SLE. (A): The circ_0005008 levels in plasma from new-onset patients with RA were significantly higher than those in healthy controls, and it was not expressed in the plasma of patients with SLE; (B): The circ_0005198 levels in plasma from new-onset patients with RA were significantly higher than those in healthy controls and patients with SLE; (C): The circ_0027089 levels in plasma were not significantly different among new-onset patients with RA and healthy controls.

FIGURE 3. Correlations between plasma circRNA_005008 and clinical variables in new-onset patients with RA (Spearman’s rank correlation coefficients). (A–D) The circ_0005008 levels in plasma from new-onset patients with RA correlated positively with DAS28, ESR, CRP, and RF. (E): The circ_0005008 levels in plasma from new-onset patients with RA showed no correlation with anti-CCP. (F–I): The plasma level of circ_0005198 in new-onset patients with RA correlated positively with DAS28, ESR, CRP, and RF. (J): The circ_0005198 levels in plasma from new-onset patients with RA showed no correlation with anti-CCP. (K,L): The circ_0005008 and circ_0005198 levels in low/moderate DAS28 group (score 2.6 to ≤5.1) were significantly lower than that in high DAS28 group (score >5.1).

FIGURE 4. ROC curves for plasma circ_0005008 and circ_0005198 in patients with RA. The AUC of plasma circ_0005008 was 0.829 (95% CI 0.754–0.904, p < 0.001) and 0.783 (95% CI 0.690–0.875, p < 0.001).

FIGURE 5. qRT-PCR determination of the relative circ_0005198 expression and its potential target miRNAs in RA-FLSs and OA-FLSs. (A): The level of circ_0005198 in OA-FLSs was significantly lower than that in RA-FLSs; (B): The level of circ_0005198 in RA-FLSs correlated with DAS28; (C,D): The level of miR-4459 and miR-1237-3p were not significantly different among RA-FLSs and OA-FLSs; (E): The RA-FLSs level of miR-4778-3p was decreased significantly compared with its OA-FLSs level; (F): The level of circ_0005198 in RA-FLSs correlated negatively with the level of miR-4778-3p.

FIGURE 6. A workflow of how plasma circ_0005008 and circ_0005198 in new-onset patients with RA might serve as potential biomarkers for disease severity and diagnosis, and how circ_0005198 might function in RA-FLSs via its interaction with miR-4778-3p.