Hyperoxia-induced methylation decreases RUNX3 in a newborn rat model of bronchopulmonary dysplasia

Abstract

BackgroundBronchopulmonary dysplasia (BPD) in premature infants is a predominantly secondary occurrence to intrauterine inflammation/infection and postpartum mechanical ventilation; in recent years, an association with epigenetics has also been found. DNA methylation, catalyzed by DNA methyl transferases (DNMTs), and tri-methylation of lysine 27 on histone H3 (H3K27me3), mediated by the methyltransferase, Enhancer of Zeste Homolog 2 (EZH2), are some of the most commonly found modifications in epigenetics. Runt-related transcription factor 3 (RUNX3) is associated with pulmonary epithelial and vascular development and regulates expression at the post-transcriptional level by DNA methylation through DNMT1 or DNMT3b. However, the involvements of these epigenetic factors in the occurrence of BPD are, as yet, unclear.MethodsNewborn rats were randomly assigned to a model, hyperoxia (85 % O2) or control, normoxia group (21 % O2). Lung tissues and alveolar type 2 (AT2) epithelial cells were collected between 1–14 days. The expression of DNMTs, and EZH2 was detected by immunohistochemistry, Western blot and real-time PCR. The percentage of DNA methylation and H3K27me3 levels in the RUNX3 promoter region was measured by bisulfite sequencing PCR and chromatin immunoprecipitation assay. RUNX3 protein and mRNA expression in AT2 cells was also measured after inhibition using the DNA methylation inhibitor, 5-Aza-2′-deoxycytidine, the H3K27me3 inhibitor, JMJD3, and the EZH2 inhibitor, DZNep.ResultsCompared with the control group, RUNX3 protein was downregulated and DNMT3b and EZH2 were highly expressed in lung tissues and AT2 cells of the model group (P < 0.05), while high DNA methylation and H3K27me3 modifications were present in the RUNX3 promoter region, in lung tissues of the model group (P < 0.05). Following hyperoxia in the model group, JMJD3 and DZNep significantly reversed the hyperoxia-induced down-regulation of RUNX3 expression in AT2 cells (P < 0.05), more so than 5-Aza-2′-deoxycytidine (P < 0.05).Conclusions1) DNA methylation and H3K27 trimethylation are present in the BPD model; 2) RUNX3 down-regulation is attributed to both DNMT3b-catalyzed DNA methylation and EZH2-catalyzed histone methylation.

Decreased ZONAB expression promotes excessive transdifferentiation of alveolar epithelial cells in hyperoxia-induced bronchopulmonary dysplasia

“…ZONAB expression may be increased in other cell types, including bronchial epithelial cells, as demonstrated by the immunofluorescence staining results. Other sources, such as fibroblasts, may have also contributed to the increased ZONAB expression in the in vivo model and are probably involved in the epithelial-mesenchymal transition (10,38), although this speculation must be confirmed in future studies. In addition, the effect of hyperoxia on endogenous and exogenous ZONAB, as well as the mechanism of how hyperoxia causes a decrease in ZONAB in AEC II are questions requiring further exploration.…”

Peptidome analysis of lung tissues from a hyperoxia‐induced bronchopulmonary dysplasia mouse model: Insights into the pathophysiological process of bronchopulmonary dysplasia

“…To understand the pathogenesis mechanism of BPD, many animal models, including newborn rodents, have been exposed to excessive levels of oxygen to induce this disease. Hyperoxia exposure in newborn rats closely mimics clinical conditions in preterm infants (Choi et al, ; Zhu et al, ). In our hyperoxia‐induced BPD mouse model, histological analysis of lung sections showed significant alveolar simplification and aberrant pulmonary vascularization (Figures b and c), which are the typical histopathological hallmarks of clinical BPD (Coalson, ; Kalikkot Thekkeveedu, Guaman, & Shivanna, ).…”

Early injury of the neonatal lung contributes to premature lung aging: a hypothesis

“…In experimental rat and mouse models of neonatal lung injury, hyperoxia was shown to result in diminished expression of histone deacetylases (HDAC) 1 and 2 similar to reduced HDAC activity in lungs of COPD patients but contrary to the increased levels of HDACs in IPF lungs [ 34 – 37 ]. In hyperoxia-treated neonatal rats, DNA methylation by DNMT3b- and EZH2-catalyzed histone methylation was observed [ 38 ]. Whether similar DNA and histone modifications also occur in chronic lung diseases remain to be investigated.…”