title: Hyperoxia-induced methylation decreases RUNX3 in a newborn rat model of bronchopulmonary dysplasia.
abstract: BACKGROUND
  Bronchopulmonary 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.
  
  
  METHODS
  Newborn 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.
  
  
  RESULTS
  Compared 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).
  
  
  CONCLUSIONS
  1) 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.
authors.affiliation: Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang, 110004, China. tingtingyuli_911@126.com.
authors.forename: Yuting
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang, 110004, China. fujh@sj-hospital.org.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Fu
authors.affiliation: Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang, 110004, China. 1789661042@qq.com.
authors.forename: Haiping
authors.initials: H
authors.lastname: Yang
authors.affiliation: Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang, 110004, China. 13840222897@qq.com.
authors.forename: Yuqing
authors.initials: Y
authors.lastname: Pan
authors.affiliation: Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang, 110004, China. yaoli7915@126.com.
authors.forename: Li
authors.initials: L
authors.lastname: Yao
authors.affiliation: Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang, 110004, China. xuexd@sj-hospital.org.
authors.forename: Xindong
authors.initials: X
authors.lastname: Xue
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001997:Bronchopulmonary Dysplasia / Q000378:metabolism* / Q000473:pathology; D050677:Core Binding Factor Alpha 3 Subunit / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D019175:DNA Methylation / Q000502:physiology*; D004195:Disease Models, Animal; D018496:Hyperoxia / Q000378:metabolism* / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D050677:Core Binding Factor Alpha 3 Subunit