Role of MicroRNA-200b in normal lung development and how it is affected in abnormal lung development associated with congenital diaphragmatic hernia

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2015-12, 2017-07, 2017-11
Khoshgoo, Naghmeh
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Annals of Surgery
Scientific Reports
Annals of Surgery
Introduction: Congenital Diaphragmatic hernia (CDH) is a developmental defect of the diaphragm associated with abnormal lung development. A common genetic cause has not been found for CDH suggesting a role of epigenetic factors including microRNAs. We recently reported the higher level of miR-200b expression in hypoplastic lungs from preterm infants with congenital diaphragmatic hernia. MicroRNA miR-200b is best known for its role in cancer and epithelial-mesenchymal transition (EMT), but our knowledge on its role during normal lung development and how it is affected in the developmental disorders of the lung is limited. We hypothesized that miR-200b is a key determinant of normal and abnormal lung development via mechanisms associated with Epithelial-Mesenchymal Transition (EMT) and Transforming Growth Factor β (TGF-β) signaling. The aim of this thesis was to delineate the role of miR-200b in lung development and lung hypoplasia in the rat model of CDH. Methodology: We used human lung tissues and biofluids to evaluate the expression of miR-200b and its targets using RT-qPCR, in situ hybridization (ISH) and immunohistochemistry (IHC). We used cultured human bronchial epithelial cells to evaluate the role of miR-200b on TGF-β/SMAD (signalling using a luciferase assay. To investigate the role of miR-200b in normal lung development, we generated a miR-200b knockout mouse (miR-200b-/-). We compared lung functions of miR-200b knockout mice to wild type mice. We determined the lung phenotype and branching using in vivo micro-computed tomography scan. To study the effect of miR-200b in hypoplastic lungs associated with CDH, we used the nitrofen rat model of lung hypoplasia and CDH. We assessed miR-200b expression using RT-qPCR and in situ hybridization. We performed loss-of-function studies to determine the effects of miR-200b on epithelial cell phenotype and function by immunostaining and scratch wound healing assay. Finally, miR-200b was used as a treatment to rescue lung hypoplasia in vivo. Results: MiR-200b abundance in the tracheal fluid from CDH fetuses was higher in the ones with a better response to a prenatal lung growth-promoting therapy. MiR-200b-/- mice displayed reduced distal airway branching, thickened alveolar walls, and less septation. They had a higher number of fibroblast-like cells resulting in significantly higher tissue damping and elastance and lower hysteresivity in the lung. In the rat model of lung hypoplasia and CDH, miR-200b expression was lower in early abnormal lung development and higher just before term when compared to control. Normalizing miR-200b abundance reduced CDH incidence by 33.6% and significantly reduced the severity of lung hypoplasia in ex vivo and in vivo model we employed. Conclusions: MiR-200b is required for normal lung branching morphogenesis and lung function. Prenatal transplacental therapy with miR-200b has the potential to prevent CDH-associated lung hypoplasia.
Congenital Diaphragmatic Hernia, miR-200b, CDH, Lung Development, Lung Hypoplasia