miR-155 in the progression of lung fibrosis in systemic sclerosis

Abstract

Background: MicroRNA (miRNA) control key elements of mRNA stability and likely contribute to the dysregulated lung gene expression observed in systemic sclerosis associated interstitial lung disease (SSc-ILD). We analyzed the miRNA gene expression of tissue and cells from patients with SSc-ILD. A chronic lung fibrotic murine model was used.

Methods: RNA was isolated from lung tissue of 12 patients with SSc-ILD and 5 controls. High-resolution computed tomography (HRCT) was performed at baseline and 2-3 years after treatment. Lung fibroblasts and peripheral blood mononuclear cells (PBMC) were isolated from healthy controls and patients with SSc-ILD. miRNA and mRNA were analyzed by microarray, quantitative polymerase chain reaction, and/or Nanostring; pathway analysis was performed by DNA Intelligent Analysis (DIANA)-miRPath v2.0 software. Wild-type and miR-155 deficient (miR-155ko) mice were exposed to bleomycin.

Results: Lung miRNA microarray data distinguished patients with SSc-ILD from healthy controls with 185 miRNA differentially expressed (q < 0.25). DIANA-miRPath revealed 57 Kyoto Encyclopedia of Genes and Genomes pathways related to the most dysregulated miRNA. miR-155 and miR-143 were strongly correlated with progression of the HRCT score. Lung fibroblasts only mildly expressed miR-155/miR-21 after several stimuli. miR-155 PBMC expression strongly correlated with lung function tests in SSc-ILD. miR-155ko mice developed milder lung fibrosis, survived longer, and weaker lung induction of several genes after bleomycin exposure compared to wild-type mice.

Conclusions: miRNA are dysregulated in the lungs and PBMC of patients with SSc-ILD. Based on mRNA-miRNA interaction analysis and pathway tools, miRNA may play a role in the progression of the disease. Our findings suggest that targeting miR-155 might provide a novel therapeutic strategy for SSc-ILD.

Keywords: Biomarker; Bleomycin; Gene expression; Lung fibrosis; Systemic Sclerosis; miR-155; microRNA.

Fig. 1

a Timeline of the study. See “Methods” for more details. b Heatmap of whole lung homogenate showing the expression of genes clustered using complete linkage hierarchical supervised clustering. Gray indicates healthy controls (HC1 to HC5; n = 5); orange indicates patients with systemic sclerosis interstitial lung disease (SSc-ILD) (SSc-A to SSc-K; n = 12). Each miRNA was z-score-normalized across all samples and was scaled to red and blue (≥2 or ≤ –2, respectively) and white indicating a z score of zero. ILD interstitial lung disease, HRCT high-resolution computer tomography, PFT pulmonary function test, FibMax HRCT lung score, FVC% percent forced vital capacity, NSIP non-specific interstitial pneumonia

Fig. 2

Whole lung homogenate with miRNA and mRNA expression by qPCR of miR-182 (a), miR-155 (b), miR-21 (c), collagen type 3 alpha-1 (Col3a1) (d), MS4A4A (e), and periostin (POSTN) (f) in healthy controls (HC) (n = 5) and patients with systemic sclerosis interstitial lung disease (SSc-ILD) (n = 14). Data are expressed as fold-change normalized to miRNA (RNU6B) or mRNA (18S) average expression in HC samples. Student’s t test; p < 0.05 was considered significant

Fig. 3

a Correlation between the delta high-resolution computerized tomography lung score (Fib _Max) and mir-155 whole lung homogenate gene expression (r = 0.65, p < 0.001). b Correlation between miR-155 expression on peripheral blood mononuclear cells by Nanostring and percentage forced vital capacity (%FVC) (r = –0.60, p = 0.01), and c percentage diffusing capacity of the lung for carbon monoxide (%DLCO) (r = –0.58, p = 0.02). d miR-21 and e miR-155 expression on lung fibroblasts from cell lines of healthy controls (HC) and patients with systemic sclerosis interstitial lung disease (SSc-ILD) stimulated for 18 hours with media, IL-13, transforming growth factor-beta (TGF-β), and interferon-alpha (IFN-α) (see “Methods”) (analysis of variance, p > 0.05 for both miRNA). Data are expressed as fold-change compared to HC samples or media control on a, b, d, and e. Data are expressed as counts of gene expression and percentage on b and c. p < 0.05 was considered significant

Fig. 4

Milder lung fibrosis in miR-155 knockout (KO) mice. a Percent survival rate by Kaplan-Meier, log-rank test. b Ashcroft lung score assessed by Masson’s trichrome staining. Lung gene expression by Nanostring of Arginase-1 (c) and tissue inhibitor of metalloproteinase-1 (TIMP1) (d). e Cluster of the most affected genes by the absence of miR-155 (blue). Data are expressed by counting gene expression on c and d. Each microRNA was z-score-normalized across all samples and scaled to red and blue (≥2 or ≤ –2, respectively) and white indicating a z score of zero. Analysis of variance; p < 0.05 was considered significant. WT wild-type, Bleo bleomycin