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Volume 3 Supplement 1

ESICM LIVES 2015

Mirna interference in human pulmonary alveolar epithelial cells (HPAEPIC) undergoing cyclic stretch and in ex vivo ventilated and perfused rat lungs

Introduction

We have previously demonstrated that the expression of miRNA 27a-5p is associated with DAD in an experimental model of ventilator-induced lung injury and in patients with ARDS.

Objectives

To modulate miRNA 27a-5p expression in HPAEPiC undergoing stretch and in ex vivo ventilated perfused lungs.

Methods

HPAEPiCs were transfected overnight with miRNA 27a-5p inhibitor (10 nM, 20 nM and 50 nM) or mimic (20 nM, 40 nM) or the negative control inhibitor (Exiqon) using HiPerfect transfection agent and underwent cyclic stretch for 6 h (15% linear elongation, 0.2 Hz). Cells were then cultured for up to 48 h for miRNA isolation (miRNeasy Mini Kit, Qiagen) and for 72 h for protein isolation (T-Per, Pierce). All assays were performed in triplicate.

Adult male Sprague-Dawley rats (weight 325-375 g) were anesthetized and sacrificed by exsanguination, and the heart and lung were extracted en bloc and mounted in a ventilation chamber for perfusion (Krebs solution) and ex vivo ventilation for 2.5 h (VT = 6 mL/kg, PEEP = 5 cm H2O (Harvard Apparatus, MA). miRNA 27a-5p inhibitor (0.25 and 0.50 mg/kg lung weight), its corresponding controls, as well as miRNA 27a-5p mimic (0.10 mg/kg) were administered intratracheally 30 min before mechanical ventilation (n = 5 for each of the 5 groups).

Expression of miRNA 27a-5p was quantified by RT-qPCR (miScript II RT and QuantiTect SYBR Green PCR, Qiagen) in a 7500 Fast Real-Time PCR (Life Technologies) and data were analyze with the ΔΔCT method. Epidermal growth factor receptor (EGFR) protein concentration, a miRNA 27a-5p target, was measured in cell and tissue lysates for Elisa.

Values were compared by the Kruskal-Wallis method. The study was conducted with the approval of the local IRB. Values are fold change as compared to the negative control, or median (IQR). p < 0.05 was considered statistically significant.

Results

Treatment with the miRNA 27a-5p inhibitor dose dependently decreased miRNA 27a-5p expression vs. negative control (10 nM: 1.05; 20 nM: 0.80; 50 nm: 0.62); and increased EGFR concentration vs negative control (10 nM: 1.20; 20 nM: 1.36; 50 nM: 1.45). Treatment with the miRNA 27a-5p mimic increased miRNA 27a-5p expression (20 nM: 150; 40 nM: 200) and decreased EGFR concentration (20 nM: 0.79; 40 nM: 0.78).

In lung tissue, miRNA 27a-5p inhibitor down-regulated miRNA 27a-5p expression (46 × 10-4 (42 × 10-4-54 × 10-4) vs. 15 × 10-4 (10 × 10-4-54 × 10-4) and 76 × 10-4 (57 × 10-4-93 × 10-4) vs. 11 × 10-4 (8 × 10-4-20 × 10-4)), for the 0.25 mg/kg and 0.50 mg/kg doses, respectively. EGFR concentration increased with the 0.50 mg/kg dose (0.17 (0.41-0.47) vs. 0.42 (0.41-0.47) pg/ug protein).

miRNA 27a-5p mimic up-regulated miRNA 27a-5p expression (3324 × 10-4 (2023 × 10-4-4825 × 10-4) vs 46 × 10-4 (42 × 10-4-54 × 10-4)) and reduced EGFR concentration (0.34 (0.22-0.43) vs. 0.11 (0.08-0.20).

Conclusions

It is possible to modulate in vitro and ex vivo the expression of miRNA 27a-5p, a miRNA associated with DAD.

Funding

Fis PI12/2898 and FEDER Funds

Author information

Correspondence to A Ferruelo.

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Keywords

  • Epidermal Growth Factor Receptor
  • Alveolar Epithelial Cell
  • Perfuse Lung
  • Krebs Solution
  • Cyclic Stretch