Analysis of miRNA Expression in Patients With NSAID-Exacerbated Respiratory Disease

Abstract

Purpose: Non-steroidal anti-inflammatory drug-exacerbated respiratory disease (N-ERD) is a phenotype of bronchial asthma that is characterized by a severe course and the presence of chronic rhinosinusitis (CRS) with nasal polyps. MicroRNAs (miRNAs) belong to a family of small, non-coding RNAs whose primary function is to regulate gene transcription. The aim of this study was to determine the miRNA profile and to validate selected miRNAs in biological material from the upper respiratory tract collected with a minimally-invasive method in patients with N-ERD.

Methods: The miRNA profile was assessed in subjects with N-ERD, CRS, and allergic asthma (AA), as well as healthy controls (HCs), using microarray technique. Following this, 6 miRNAs were validated using reverse transcription polymerase chain reaction in 77 subjects.

Results: The profiling identified 23 miRNAs whose expression significantly differed between patients with N-ERD and HCs. Based on these results, 6 miRNAs were selected for further validation. It was found that patients with N-ERD had significantly different expressions of miR-34a-5p and miR-22-5p compared to those with AA. In the whole study group, significant correlations were found between miR-7d-3p/miR-34a-5p/miR-22-5p and the presence of blood eosinophilia (r = 0.25, r = 0.28 and r = 0.26, for all P < 0.05). Forced expiratory volume in 1 second/forced vital capacity was correlated with miR-149a-5p expression (r = 0.27, P < 0.05).

Conclusions: The results indicate that the miRNA profile in nasal mucosal lining fluid of patients with N-ERD differs from patients with AA, CRS, and compared to HCs. Some of the miRNAs selected on the basis of profiling may be involved in the regulation of eosinophilic inflammation in the respiratory tract. Our findings suggest that specific miRNAs may be considered as potential biomarkers of N-ERD.

Keywords: MicroRNAs; asthma; biomarker; drug hypersensitivity; eosinophils; nasal mucosa.

Fig. 1. A diagram describing the study design.

miRNA, microRNA; NLF, nasal lining fluid; qPCR, quantitative polymerase chain reaction; RT-PCR, reverse transcription polymerase chain reaction.

Fig. 2. Venn diagram with miRNAs unique and common for N-ERD (n = 5), AA (n = 5) and CRS (n = 5) compared to controls (n = 5). miRNAs showing more than a 2-fold up-regulation (P < 0.05) are marked with green; miRNAs showing more than 2-fold down-regulation (P < 0.05) are marked with red.

N-ERD, non-steroidal anti-inflammatory drug exacerbated respiratory disease; AA, allergic asthma; CRS, chronic rhinosinusitis; miRNA, microRNA.

Fig. 3. Venn diagram with miRNAs unique and common for AA (n = 5), CRS (n = 5) and controls (n = 5) compared to N-ERD (n = 5). miRNAs showing more than a 2-fold up-regulation (P < 0.05) are marked with green; miRNAs showing more than 2-fold down-regulation (P < 0.05) are marked with red. miRNAs selected for validation are circled.

AA, allergic asthma; CRS, chronic rhinosinusitis; N-ERD, non-steroidal anti-inflammatory drug exacerbated respiratory disease; miRNA, microRNA.

Fig. 4. Differences in expression (relative ratio) of miRNAs between patients with N-ERD/CRS/AA and controls. Comparison between groups was made with the Mann-Whitney test. Boxes: 25%–75% percentile; bars: median value; whiskers: non-outliers range.

miRNA, microRNA; N-ERD, non-steroidal anti-inflammatory drug exacerbated respiratory disease; CRS, chronic rhinosinusitis; AA, allergic asthma.

Fig. 5. Correlations between miRNA expression and clinical parameters in the study patients. (A) Correlation between miRNA expressions and blood eosinophilia. (B) Correlation between miRNA expression and forced expiratory volume in the first one second to the forced vital capacity. Correlation was performed with the use of Spearman’s rank correlation test.

FEV1/FVC%, forced expiratory volume in 1 second to the forced vital capacity ratio; Eos abs., eosinophilia absolute; miRNA, microRNA. Include exact P value in the Fig. 5A.