The role of the RNA-binding protein Quaking in lung development and congenital diaphragmatic hernia
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Date
2019-04-01
Authors
Ameis, Dustin
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Abstract
Introduction: Every 10 minutes a baby is born with congenital diaphragmatic hernia (CDH) worldwide. These babies have a hole in their diaphragm and abdominal organs in their chest, causing abnormal lung development and breathing problems and mortality at birth. The microRNA miR-200b promotes normal lung development in the rat model of CDH (Nitrofen model), possibly through repressing the RNA-binding protein Quaking (QK). I hypothesize that QK plays a key role in mediating lung development and is dysregulated in the rat Nitrofen lungs.
Methods: QK protein expression in normal prenatal rat lungs was assessed at gestational days (E) 13, 15, 18, and 21 with immunostaining. Pregnant rats were administered Nitrofen to induce CDH in the fetuses. QK expression in E21 Nitrofen lungs were compared to control using immunostaining and RT-qPCR. Normal E13 rat lungs were cultured ex vivo at an air-liquid interface. QK translation was blocked using a vivo-morpholino, and knockdown was verified with immunostaining (quantified with ImageJ). Lung branching was checked daily over four days of culture and later immunostained. An RT-qPCR panel was used to assess changes in cell type abundances in the lung explants.
Results: Immunostaining showed QK protein expression in every lung cell across the developmental stages studied. QK mRNA was increased 2.2-fold in the Nitrofen group (P < 0.05). However, protein levels were unchanged. The vivo-morpholino induced 50% QK protein knockdown (P < 0.05), resulting in fewer branches and a more diffuse periphery in the lung explants. Multiciliated cells were increased 2-fold in abundance and basal cell numbers were decreased 2-fold, implied by changes in the mRNA of cilia marker Foxj1 (P < 0.05) and the basal marker P63 (P < 0.05). Foxj1+ cells increased 2.4-fold in number according to immunostaining. IL-6 mRNA was increased 4.7-fold (P = 0.6).
Conclusion: QK is widely expressed during lung development. The upregulation of QK mRNA, not protein, in the Nitrofen group implies translation is “stalled.” However, the implications for CDH remain unclear. IL-6 has been previously shown to promote ciliogenesis through basal cell differentiation in the lungs, indicating that QK is a negative regulator of this pathway.
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Keywords
Quaking, Lung explant, Lung development, RNA-binding protein, Interactomics, RT-qPCR, Immunostaining, Basal cell, IL-6, Congenital diaphragmatic hernia, miR-200b, Multiciliated cell