Identification of circular RNAs hsa_circ_0044235 and hsa_circ_0068367 as novel biomarkers for systemic lupus erythematosus

Abstract

Circular RNAs (circRNAs) have emerged as novel biomarkers for disease diagnosis. However, the expression profiles and clinical significance of circRNAs in peripheral blood mononuclear cells (PBMCs) from systemic lupus erythematosus (SLE) remain unclear. In the present study, the expression profile of circRNAs in PBMCs from patients with SLE and healthy controls (HCs) was detected by using microarray analysis and verified by reverse transcription‑quantitative polymerase chain reaction. A total of 1,603 circRNAs were identified to be significantly aberrantly expressed in PBMCs from patients with SLE. Validation assays in 30 SLE patients and 20 HCs demonstrated that the levels of hsa_circ_0044235 and hsa_circ_0068367 were significantly decreased in the patients with SLE. Receiver operating characteristic curve analysis suggested that hsa_circ_0044235 and hsa_circ_0068367 were significant for SLE diagnosis. Furthermore, the diagnostic potential of hsa_circ_0044235 and hsa_circ_0068367 for SLE was validated in an independent validation set with 45 patients with SLE, 38 HCs and 30 patients with rheumatoid arthritis. In addition, the level of hsa_circ_0044235 in the PBMCs from patients with SLE were identified to be significantly increased in new‑onset SLE patients and in patients who were determined to be positive for anti‑double‑stranded DNA and anti‑ribosomal protein P antibodies. Additionally, the level of a microRNA (miRNA) target of hsa_circ_0044235, hsa‑miRNA‑892a, was identified to be significantly increased in the PBMCs from patients with SLE. The present study suggested that the dysregulation of circRNAs may serve a role in SLE pathogenesis, and that the levels of hsa_circ_0044235 and hsa_circ_0068367 in PBMC have potential as biomarkers for SLE diagnosis.

Figure 1

Determination of the circRNA expression profiles in PBMCs from 4 SLE patients and 4 HCs by microarray analysis. (A) Box plot visualization of the distributions of a dataset for the circRNAs profiles. (B) CircRNA scatter plot. Dots above the top green line and below the bottom green line indicate >1.5-fold changes in log-transformed circRNA data between the two compared groups. (C) Heatmap of differentially expressed circRNAs. circRNAs, circular RNAs; HCs, healthy controls; PBMCs, peripheral blood mononuclear cells; SLE, systemic lupus erythematosus.

Figure 2

Determination of the relative expression levels of circRNAs in the training set in PBMCs from 30 patients with SLE and 20 HCs by reverse transcription quantitative polymerase chain reaction. (A) The average expression levels of hsa_circ_0044235 in the PBMCs of patients with SLE were significantly decreased compared with those of the HCs. (B) The average expression levels of hsa_circ_0068367 in the PBMCs of patients with SLE were significantly decreased compared with those of the HCs. (C) The expression of hsa_circ_0037274 did not exhibit any significant differences between patients with SLE and the HCs. circRNAs, circular RNAs; HCs, healthy controls; PBMCs, peripheral blood mononuclear cells; SLE, systemic lupus erythematosus.

Figure 3

Receiver operating characteristic analysis of confirmed circRNAs in peripheral blood mononuclear cells from patients with SLE. (A) The largest AUC was identified for hsa_circ_0042345, followed by hsa_circ_0068367. (B) The AUC of combined circRNAs (hsa_circ_0042345 + hsa_circ_0068367) was 0.876. CircRNAs, circular RNAs; SLE, systemic lupus erythematosus; AUC, area under the curve.

Figure 4

Double validation of the diagnostic value of differentially expressed PBMCs circRNAs. (A) The expression level of hsa_circ_0044235 was markedly decreased in the patients with SLE compared with patients with RA and HCs. (B) The expression level of hsa_circ_0068367 were markedly decreased in the patients with SLE compared with patients with RA and HCs. (C) ROC curve analysis of hsa_circ_0042345 and hsa_circ_0068367 in patients with SLE compared with HCs. (D) ROC curve analysis of hsa_circ_0042345 and hsa_circ_0068367 for the risk-score in SLE patients compared with all controls (HCs and RA). (E) ROC curve analysis of hsa_circ_0042345 and hsa_circ_0068367 for the risk-score in patients with SLE vs. RA. PBMCs, peripheral blood mononuclear cells; CircRNAs, circular RNAs; SLE, systemic lupus erythematosus; RA, rheumatoid arthritis; HCs, healthy controls; ROC, receiver operating characteristic.

Figure 5

Correlations between the expression of hsa_circ_0044235 with autoantibodies. (A) The levels of hsa_circ_0044235 were significantly increased in patients who were positive for anti-dsDNA antibodies compared with patients who were negative for anti-dsDNA antibodies in the double validation testing set. (B) The levels of hsa_circ_0044235 was significantly increased in patients who were positive for anti-RIB-P antibodies compared with patients who were negative for anti-RIB-P antibodies in the double validation testing set. (C) The levels of hsa_circ_0044235 were significantly increased in patients who were positive for anti-dsDNA antibodies positive compared with patients who were negative for anti-dsDNA antibodies in the double validation and validation testing sets. (D) The levels of hsa_circ_0044235 were significantly increased in patients who were positive for anti-RIB-P antibodies compared with patients who were negative for anti-RIB-P antibodies in the double validation and validation testing sets. Anti-dsDNA, anti-double-stranded DNA; RIB-P, ribosomal protein P.

Figure 6

Comparison of the levels of circRNA (hsa_circ_0044235 and hsa_circ_0068367) between patients with new-onset and relapsed SLE conditions. (A) The level of hsa_circ_0044235 was significantly increased in patients with new-onset disease. (B) The level of hsa_circ_0068367 appeared to be increased in the patients with new-onset disease, but the difference was not significant. circRNA, circular RNA; SLE, systemic lupus erythematosus.

Figure 7

Comparison of the levels of miRNAs in PBMCs between patients with SLE and HCs. (A) The level of hsa-miR-135a-5p was not markedly different between patients with SLE and HCs. (B) The level of hsa-miR-135b-5p was not markedly different between patients with SLE and HCs. (C) The level of hsa-miR-892a was increased significantly in the PBMCs from patients with SLE compared with the HCs. (D) The level of hsa-miR-136-5p was decreased significantly in the PBMCs from patients with SLE compared with the HCs. (E) The level of hsa-miR-501-5p was not markedly different between the patients with SLE and HCs. miRNAs/miR, microRNAs; PBMCs, peripheral blood mononuclear cells; SLE, systemic lupus erythematosus; HCs, healthy controls.