MicroRNA Expression Profiling in Behçet's Disease

Abstract

Background: Behçet's disease (BD) is a chronic inflammatory multisystem disease characterized by oral and genital ulcers, uveitis, and skin lesions. MicroRNAs (miRNAs) are key regulators of immune responses. Differential expression of miRNAs has been reported in several inflammatory autoimmune diseases; however, their role in BD is not fully elucidated. We aimed to identify miRNA expression signatures associated with BD and to investigate their potential implication in the disease pathogenesis.

Methods: miRNA microarray analysis was performed in blood cells of BD patients and healthy controls. miRNA expression profiles were analyzed using Affymetrix arrays with a comprehensive coverage of miRNA sequences. Pathway analyses were performed, and the global miRNA profiling was combined with transcriptoma data in BD. Deregulation of selected miRNAs was validated by real-time PCR.

Results: We identified specific miRNA signatures associated with BD patients with active disease. These miRNAs target pathways relevant in BD, such as TNF, IFN gamma, and VEGF-VEGFR signaling cascades. Network analysis revealed several miRNAs regulating highly connected genes within the BD transcriptoma.

Conclusions: The combined analysis of deregulated miRNAs and BD transcriptome sheds light on some epigenetic aspects of BD identifying specific miRNAs, which may represent promising candidates as biomarkers and/or for the design of novel therapeutic strategies in BD.

Figure 1

Validation of differentially expressed miRNAs by real-time PCR. Real-time PCR for the indicated miRNAs were performed in healthy controls (Healthy) and in BD patients (Behçet). Values are calculated as fold change with respect to healthy samples with the ΔΔCt method. miR-16-5p and miR-26-5p were used as endogenous controls for miRNA expression (see Material and Methods). Histograms indicate mean values; bars indicate standard deviation (SD). p = 0.05 (Mann–Whitney test).

Figure 2

Histogram representing pathways enriched in BD-modulated miRNA targets genes and in BD DEGs. y axis: −log₁₀(p value) (hypergeometric test).

Figure 3

Selection of miRNAs that targeted genes differentially expressed in BD. (a) Pie chart showing the percentage of BD DEGs that were targeted by miRNAs modulated in BD. (b) BD-modulated miRNAs and their respective targeted BD DEGs. Genes associated with Th17 cells are written in bold characters.

Figure 4

Functional classification of BD DEGs targeted by selected miRNAs. Pie charts showing the different GO biological processes in which BD DEGs targeted by selected deregulated miRNAs in BD can be classified.

Figure 5

Network analysis of genes modulated in Behçet's disease. (a) PPI network of differentially expressed genes in BD. (b–g) Graphical representation of the six clusters that were extracted from the PPI network of modulated genes in BD. Red dots indicate DEGs that are targeted by modulated miRNAs in BD.