Involvement of miRNA-34a regulated Krüppel-like factor 4 expression in hyperoxia-induced senescence in lung epithelial cells

Abstract

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% O2/5% CO2) or air (21% O2/5% CO2) for 24 h. Newborn mice (< 12 h old) were exposed to hyperoxia (> 95% O2) 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.

Emerging role of cellular senescence in normal lung development and perinatal lung injury

“…Although type II alveolar cells account for a very small percentile of senescent cells, these cells contribute to hyperoxic lung injury via paracrine SASP factors. 7 , 39 In fact, the majority (92%) of 5-dodecanoylaminofluorescein di- β -D-galactopyranoside (C 12 FDG; a substrate of SA- β -gal) positive cells were lung macrophages. These macrophages also highly express the senescence biomarkers B2M (encoding β 2-microglobulin) 40 and Plaur (encoding uPAR) 10 as well as SASP factors, further confirming that these macrophages are senescent.…”

“… 53 We recently reported that miRNA-34a contributes to hyperoxia-induced senescence in cultured lung epithelial cells. 39 Nevertheless, further study is warranted to investigate whether these signaling pathways participate in the development of alveolar and vascular simplification seen in bronchopulmonary dysplasia by modulating cellular senescence.…”

Mechanism and role of nuclear laminin B1 in cell senescence and malignant tumors

“…Li et al [ 33 ] studied the damages caused by ethanol in human vascular diseases; the results showed that doxycycline restored the level of aging-related proteins such as LMNB1 by reducing the extent of mTOR and NF-κB activation, thus alleviating ethanol-induced inflammation and aging. By observing the changes of mouse lung epithelial cells and primary human small airway epithelial cells after treatment with a high concentration of oxygen (95% O 2 /5% CO 2 ) or air (21% O 2 /5% CO 2 ) for 24 h. Maeda et al [ 34 ] found that high oxygen concentration induced senescence by increasing the level of miR-34a-5p. Moreover, LMNB1 gene deletion increased p21 gene expression.…”