MicroRNA 219-5p inhibits alveolarization by reducing platelet derived growth factor receptor-alpha

Abstract

Background: MicroRNA (miR) are small conserved RNA that regulate gene expression post-transcription. Previous genome-wide analysis studies in preterm infants indicate that pathways of miR 219-5p are important in infants with Bronchopulmonary Dysplasia (BPD).

Methods: Here we report a prospective cohort study of extremely preterm neonates wherein infants diagnosed with severe BPD expressed increased airway miR-219-5p and decreased platelet derived growth factor receptor alpha (PDGFR-α), a target of mir-219-5p and a key regulator of alveolarization, compared to post-conception age-matched term infants.

Results: miR-219-5p was highly expressed in the pulmonary epithelial lining in lungs of infants with BPD by in situ hybridization of human infant lungs. In both in vitro and in vivo (mouse) models of BPD, miR-219-5p was increased on exposure to hyperoxia compared with the normoxia control, with a complementary decrease of PDGFR-α. To further confirm the target relationship between miR-219 and PDGFR-α, pulmonary epithelial cells (MLE12) and lung primary fibroblasts were treated with a mimic of miR-219-5p and a locked nucleic acid (LNA) based inhibitor of miR-219-5p. In comparison with the control group, the level of miR-219 increased significantly after miR-219 mimic treatment, while the level of PDGFR-α declined markedly. LNA exposure increased PDGFR-α. Moreover, in BPD mouse model, over-expression of miR-219-5p inhibited alveolar development, indicated by larger alveolar spaces accompanied by reduced septation.

Conclusions: Taken together, our results demonstrate that increased miR-219-5p contributes to the pathogenesis of BPD by targeting and reducing PDGFR-α. The use of specific miRNA antagonists may be a therapeutic strategy for preventing the development of BPD.

Keywords: Bronchopulmonary dysplasia; Infant; Lung development; microRNAs.

Fig. 1

Airway miR-219-5p (miR 219) and PDGFR-α for BPD. 30 human tracheal aspirate samples collected from full term and BPD infants were analyzed for miR-219 (a) by qPCR and PDGFR-α (b) by ELISA. Values are means ± SE; n = 30 samples /group. *P ˂ 0.05 vs. corresponding group

Fig. 2

Expression of miR-219-5p (miR 219) is increased in human bronchopulmonary dysplasia (BPD), compared with term lung of comparable gestational age. a, b: In situ hybridization of miR-219 in FFPE sections of human BPD and normal lungs. c: miR-219 expression by qPCR in RNA isolated from human lung samples of full term lung and BPD lung at 36–40 wk postmenstrual age (PMA). Values are means ± SE; n = 4 samples/group. *P ˂ 0.05 vs. other groups

Fig. 3

Expression of miR-219-5p (miR-219) and PDGFR-α in MLE12 and primary lung fibroblasts exposed to air or hyperoxia and treated with either mimic miR-219 or locked nucleic acid (LNA)-miR-219 inhibitor. a, b: miR-219 was increased while PDGFR-α was decreased in MLE12 and primary lung fibroblasts exposed to hyperoxia for 24 h, compared with normoxia. c, d Representative graphs showing significant decrease of PDGFR-α in mimic miR-219 transfected group and increase of PDGFR-α in LNA-miR-219 inhibitor transfected group compared to its control in both MLE12 and primary lung fibroblasts. Values are means ± SE; *P ˂ 0.05 vs. corresponding group

Fig. 4

miR-219-5p (miR-219) and PDGFR-α during lung development and effect of hyperoxia and mimic miR-219 on miR-219 in a BPD mouse model. a Expression of miR-219 in lung homogenates from mice at 4, 7, and 14 days of age (P1, P14, and P42). b Expression of PDGFR-α by quantitative RT-PCR (qPCR) at P1, P14, and P42, showing increase PDGFR-α at P14, and P42 when miR-219 is decreased. Lung homogenates from mice at 14 days of age exposed to air or hyperoxia were analyzed by qPCR of RNA from lung homogenates for miR-219 (c) and PDGFR-α (d) or by western blot for PDGFR-α (e). f Representative photomicrographs of H&E-stained sections of alveolar regions from lungs of mice at 14 days of age administered either with mimic miR-219, or control mimic. g Radial alveolar counts (RAC) of mice administered control vs mimic miR-219 in normoxia and hyperoxia. Values are means ± SE; n = 6 mice/group. *P ˂ 0.05 vs. corresponding group