The role of miR-431-5p in regulating pulmonary surfactant expression in vitro

Pulmonary surfactant is the complex mixture of lipid and protein that covers the alveolar surface. Pulmonary surfactant deficiency is one of the main causes of neonatal respiratory distress. Recent studies showed that miRNA plays an important role in lung development, but research into miR-431 regulation of pulmonary surfactant are sparse. In this study, we explored the regulatory role of miR-431-5p in the expression of pulmonary surfactant and identified its potential target gene, Smad4.

The bioinformatics tool TargetScan was used to predict the targets of miR-431. The expression of miR-431-5p was achieved via transfection of miR-431-5p mimics, an miR-431-5p inhibitor and corresponding negative control. The level of miR-431-5p was determined using quantitative real-time PCR. The CCK8 assay was conducted to confirm cell growth 12 h after transfection with miR-431-5p mimics, inhibitor or NC. Smad4 and surfactant-associated proteins in A549 were analyzed using western blot and quantitative real-time PCR.

Smad4 was validated as a target of miR-431 in A549 cells. Overexpression of miR-431 accelerated A549 proliferation and inhibited A549 apoptosis. The mRNA and protein levels for the surfactant proteins (SP-A, SP-B, SP-C and SP-D) were found to be differentially expressed in A549 cells over- or under-expressing miR-431-5p.

Our results show that miR-431-5p is critical for pulmonary surfactant expression and that its regulation is closely related to the TGF-β/Smad4 pathway. These results will help us to study the pathophysiological mechanism of lung developmental diseases.