Involvement of MiRNA-34a Regulated Krüppel-like Factor 4 Expression in Hyperoxia-induced Senescence in Lung Epithelial Cells

Background: Premature infants, subjected to supplemental oxygen and mechanical ventilation, may develop bronchopulmonary dysplasia, a chronic lung disease characterized by alveolar dysplasia and impaired vascularization. We and others have shown that hyperoxia causes senescence in cultured lung epithelial cells and fibroblasts. Although miR-34a modulates senescence, it is unclear whether it contributes to hyperoxia-induced senescence. We hypothesized that hyperoxia increases miR-34a levels, leading to cellular senescence.

Methods: We exposed mouse lung epithelial (MLE-12) cells and primary human small airway epithelial cells to hyperoxia (95% O/5% CO) or air (21% O/5% CO) for 24 h. Newborn mice (< 12 h old) were exposed to hyperoxia (> 95% O) for 3 days and allowed to recover in room air until postnatal day 7. Lung samples from premature human infants requiring mechanical ventilation and control subjects who were not mechanically ventilated were employed.

Results: Hyperoxia caused senescence as indicated by loss of nuclear lamin B1, increased p21 gene expression, and senescence-associated secretory phenotype factors. Expression of miR-34a-5p was increased in epithelial cells and newborn mice exposed to hyperoxia, and in premature infants requiring mechanical ventilation. Transfection with a miR-34a-5p inhibitor reduced hyperoxia-induced senescence in MLE-12 cells. Additionally, hyperoxia increased protein levels of the oncogene and tumor-suppressor Krüppel-like factor 4 (KLF4), which were inhibited by a miR-34a-5p inhibitor. Furthermore, KLF4 knockdown by siRNA transfection reduced hyperoxia-induced senescence.

Conclusion: Hyperoxia increases miR-34a-5p, leading to senescence in lung epithelial cells. This is dictated in part by upregulation of KLF4 signaling. Therefore, inhibiting hyperoxia-induced senescence via miR-34a-5p or KLF4 suppression may provide a novel therapeutic strategy to mitigate the detrimental consequences of hyperoxia in the neonatal lung.