Mechanical stretch differentially regulates the expression of specific miRNA in the extracellular vesicles released from lung epithelial cells

Mechanical force is critical for lung development. In this study we identified specific EV-miRNAs released from the mouse epithelial cell line in response to mechanical stretch involve in lung development. In utero fetal lung experiences significant continuous transpulmonary pressure as a result of epithelial secretion in to the airway lumen, and periodic fetal breathing movement that move the fluid along the developing airway. Mechanical force is important factors for fetal lung development. However, the effect of mechanical force on the functions of lung cells is not known precisely. Extracellular vesicles –microRNAs (EV-miRNA) are increasingly recognized as a new mode of cell-to-cell communication. miRNA is well known as a regulator of physio-pathological process. In this study, we used oligonucleotide microarray technology to investigate miRNA expression in EV-released from mouse lung epithelial cell MLE12 after exposed to 10% cyclic or 5% continuous stretch. Analysis of microarray data identified 9 and 33 miRNAs significantly differentially expressed by the cyclic and continuous stretch respectively. Several differentially expressed miRNAs were reported dynamically expressed in mouse developing lung. miRNAs associated with important transcription factors for cell function and key signaling pathways for fetal lung development also identified in this study. We conclude that mechanical signals differentially regulate the expression of specific EV/miRNAs in MLE12 are important for intercellular communication during lung fetal development. MLE12 cell line was obtained from Americal Type Culture Collection (Manassas, Virginia) expresses features of type II lung epithelial cells. Cells were culture in bioflex multiwall plates precoated with collagen-1 for 24 h, after fresh medium was replaced, plates were mounted in Flexcell FX-4000 Stretch Unit to expose 10% cyclic or 5% continuous stretch for 24 h. Parallel, cells grown on non-stretched membrane were used as control. Cell culture media (250 ml) from 175 x 106 cells were used to isolate EVs for a single experiment. The Flexcell Strain Unit has capacity to culture approximately 35 x 106 cells for experiment, a combination of 5 independent experiments were combined to take single experiment.