Impact of Diminished Expression of circRNA on Multiple Sclerosis Pathomechanisms

Abstract

Circular RNA (circRNA) molecules represent a novel and unique class of endogenous non-coding RNAs controlling the expression and function of microRNA (miRNA) and post-transcriptional regulation. Recent studies implicated circRNA in the pathomechanism of multiple sclerosis (MS). Hybridization microarray was used to define the circRNA profile in the peripheral blood mononuclear cells (PBMCs) from 20 untreated patients with relapsing-remitting MS (RRMS: 10 in relapse, 10 in remission) and 10 healthy controls (HCs). We analyzed close to 14,000 individual circRNAs per sample. The discovery set data were validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) with an independent cohort of 45 RRMS patients (18 in relapse, 27 in remission) and 27 HCs. Microarray analysis revealed 246 circRNAs differentially downregulated (P < 0.05) in RRMS patients versus HCs. We validated two circRNAs of the three showing the lowest levels of differential expression in the RRMS remission group versus the HC group: hsa_circRNA_101145 and hsa_circRNA_001896. Their expression was significantly decreased during remission in RRMS (P = 0.0000332, FC = 0.385 and P = 0.0455, FC = 0.591, respectively) and in patients showing a lower level of disability (hsa_circRNA_101145, P = 0.0695; hsa_circRNA_001896, P = 0.0008). Bioinformatic analysis revealed 10 miRNAs interacting with these circRNAs in a complementary manner and led to the discovery of three protein-coding mRNAs downregulated in patients with RRMS during remission. These transcripts have been previously implicated in oxidative stress, blood-brain barrier permeability, microglia function, and extracellular matrix molecules altering the microenvironment and inhibiting oligodendrocyte progenitor cells. circRNAs displayed a distinct profile in PBMCs from patients with RRMS, and our results may implicate circRNAs with low expression in important mechanistic pathways of RRMS.

Keywords: RNA–RNA interactions; biomarkers; circular RNA; multiple sclerosis; non-coding RNA.

Figure 1

The heatmap shows differentially downregulated circular RNAs (circRNAs) from unsupervised hierarchical clustering present in the peripheral blood mononuclear cells (PBMCs) of patients with relapsing–remitting multiple sclerosis (RRMS) in the remission group versus the relapse group versus controls.

Figure 2

Validation of the differential downregulation of the selected circRNAs in RRMS. (A) The expression levels of the indicated circRNAs were analyzed via qRT-PCR. Significance was determined by analysis of variance (ANOVA) with the Scheffé post-hoc test. *P < 0.05, ***P < 0.0005. The dotted line represents the mean value ( ± SD). (B) Receiver operating characteristic (ROC) curves for the comparison of hsa_circ_101145, hsa_circ_001896, and hsa_circ_100772 expression levels in RRMS relapse versus control, RRMS remission versus control, and RRMS relapse versus remission.

Figure 3

circRNA expression in PBMCs in relation to magnetic resonance imaging activity in RRMS. Box and whisker plots of the relative expression of the indicated circRNAs in PBMCs in gadolinium-enhanced (Gd)+ (n = 8) and in (Gd)− (n = 13) RRMS patients. Significance was determined by Student’s t-test.

Figure 4

Correlation between downregulated circRNA levels with clinical RRMS parameters. (A) PBMC hsa_circRNA_101145, hsa_circRNA_001896, and hsa_circRNA_100772 expression levels were plotted against disease duration. Circles represent individual data, and the line represents a correlation trend; P-values were calculated by R ² statistic. (B) PBMC hsa_circRNA_101145, hsa_circRNA_001896, and hsa_circRNA_100772 expression levels were plotted against EDSS. Circles represent individual data, and the line represents a correlation trend; P-values were calculated by R ² statistics.

Figure 5

mRNA predicted from miRNA identified as interacting with circRNA. (A) The Venn diagrams display the overlap of the mRNA predicted from the hsa_circRNA_101145-regulated miRNA (hsa-miR-153-5p, hsa-miR-7-5p, hsa-miR-493-3p, hsa-miR-205-3p, hsa-miR-181c-5p) and hsa_circRNA_001896-regulated miRNA (hsa-miR-329-5p, hsa-miR-26a-2-3p, hsa-miR-136-5p, hsa-miR-153-5p, hsa-miR-26a-1-3p). The figures indicate the number of overlapping mRNAs regulated by the corresponding miRNAs. (B) The Venn diagram of the overlapping mRNA regulated by all miRNAs predicted to interact with hsa_circRNA_101145 and hsa_circRNA_001896. Figures indicate the number of overlapping mRNAs regulated by all miRNAs predicted from a single circRNA.