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

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.


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 (miR-Neasy 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.