Pulmonary microRNA profiling in a mouse model of ventilator-induced lung injury

Abstract

The aim of this study was to investigate the changes induced by high tidal volume ventilation (HVTV) in pulmonary expression of micro-RNAs (miRNAs) and identify potential target genes and corresponding miRNA-gene networks. Using a real-time RT-PCR-based array in RNA samples from lungs of mice subjected to HVTV for 1 or 4 h and control mice, we identified 65 miRNAs whose expression changed more than twofold upon HVTV. An inflammatory and a TGF-β-signaling miRNA-gene network were identified by in silico pathway analysis being at highest statistical significance (P = 10(-43) and P = 10(-28), respectively). In the inflammatory network, IL-6 and SOCS-1, regulated by miRNAs let-7 and miR-155, respectively, appeared as central nodes. In TGF-β-signaling network, SMAD-4, regulated by miR-146, appeared as a central node. The contribution of miRNAs to the development of lung injury was evaluated in mice subjected to HVTV treated with a precursor or antagonist of miR-21, a miRNA highly upregulated by HVTV. Lung compliance was preserved only in mice treated with anti-miR-21 but not in mice treated with pre-miR-21 or negative-control miRNA. Both alveolar-arterial oxygen difference and protein levels in bronchoalveolar lavage were lower in mice treated with anti-miR-21 than in mice treated with pre-miR-21 or negative-control miRNA (D(A-a): 66 ± 27 vs. 131 ± 22, 144 ± 10 mmHg, respectively, P < 0.001; protein concentration: 1.1 ± 0.2 vs. 2.3 ± 1, 2.1 ± 0.4 mg/ml, respectively, P < 0.01). Our results show that HVTV induces changes in miRNA expression in mouse lungs. Modulation of miRNA expression can affect the development of HVTV-induced lung injury.

Fig. 1.

MicroRNA/gene networks induced by mechanical ventilation with high tidal volume (HVTV) in mice. A: inflammatory signaling microRNA-gene network. B: TGF-β signaling microRNA-gene network. Lines indicate the predicted interactions between microRNAs and their downstream targets. TLR4, Toll-like-receptor 4; TNF, tumor-necrosis-factor; JAK2, Janus kinase 2; SOCS1, suppressor of cytokine signaling 1; IL1A, interleukin 1A; IL6, interleukin 6; TRAF6, TNF receptor-associated factor 6; IRAK1, interleukin-1 receptor-associated kinase 1; NFAT5, nuclear factor of activated T-cells 5; IL10, interleukin 10; TGFBR2, transforming growth factor β receptor type II; IRS2, insulin receptor substrate 2; CDH1, cadherin 1; ITGA6, integrin α6; BMPR2, bone morphogenetic protein receptor, type II; EP300, E1A binding protein p300; RHOB, ras homolog gene family, member B; FOXO1, forkhead box O1; E2F3, E2F transcription factor 3; CTNNA1, catenin (cadherin-associated protein) α1; RUNX3, runt-related transcription factor 3.

Fig. 2.

Changes in the levels of mRNAs encoding IL-6, SOCS1, and SMAD4 in lungs of mice exposed to mechanical HVTV for 1 or 4 h compared with control mice: *P < 0.05 vs. control.

Fig. 3.

In situ hybridization with double DIG-LNA miR-21 probe, anti-DIG-AP antibody, and AP solution containing NBT-BCIP (purple staining) of formalin-fixed, paraffin-embedded lung sections, from mice subjected for 4 h to HVTV (a, c, e, g, i), and control mice (b, d, f, h, j). MiR-21 staining is observed in lungs from mice subjected to HVTV in alveolar type II pneumocytes (a and b), bronchiole lining cells (c and d), endothelial cells (e and f), myofibroblasts (g and h), as well as in alveolar macrophages (i and j; arrows indicating specific cell type; a, b, c, d, e, h, i, j: magnification ×400, f and g: magnification ×600).

Fig. 4.

A: changes in levels of miR-21 in lung homogenates from mice treated with negative control miR, anti-miR-21, or pre-miR-21 followed by mechanical HVTV for 4 h, compared with control mice: *P < 0.05 for all ventilated mice compared with control mice, #P < 0.05 for pre-miR-21 treatment vs. negative control miR, ^P < 0.05 for anti-miR-21 treatment vs. negative control miR. B: changes in levels of BMPR2 mRNA in lung homogenates from mice treated with negative control miR, anti-miR-21, or pre-miR-21 followed by HVTV for 4 h, compared with control mice: *P < 0.001 for negative-control-miR and pre-miR-21 treatment vs. control, and PTEN mRNA; *P < 0.001 for all ventilated mice vs. control, #P < 0.05 for anti-miR-21 treatment vs. negative-control-miR and pre-miR-21.

Fig. 5.

Evaluation of lung injury in mice treated with negative control miR, anti-miR-21, or pre-miR-21 followed by mechanical HVTV for 4 h, compared with control mice. All data are presented in whisker blots (min-max, line at mean). A: inspiratory capacity as % control: *P < 0.001 for negative-control-miR and pre-miR-21 treatment vs. control. B: bronchoalveolar lavage fluid (BALF) protein concentration: *P < 0.001 for control vs. all ventilated mice, #P < 0.05 for anti-miR-21 treatment vs. negative-control-miR and pre-miR-21. C: BALF concentrations of IL-6: *P < 0.001 for control vs. all ventilated mice. D: BALF concentrations of macrophage inflammatory protein (MIP)-2: *P < 0.001 for control vs. all ventilated mice, #P < 0.05 for anti-miR-21 vs. pre-miR-21 treatment.