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

Respiratory Research

Xindong Xue¹  Li Yao¹  Yuqing Pan¹  Haiping Yang¹  Jianhua Fu¹  Yuting Zhu¹ 

[1] Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang 110004, China

关键词: bronchopulmonary dysplasia, EZH2;    alveolar development;    RUNX3;    histone methylation;    DNA methylation;   

Others  :  1233542
DOI  :  10.1186/s12931-015-0239-x

 received in 2015-01-10, accepted in 2015-06-16,  发布年份 2015

【 摘 要 】

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 % O ₂ ) or control, normoxia group (21 % O ₂ ). 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.

【 授权许可】

   
2015 Zhu et al.

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【 参考文献 】

• [1]Bhandari A, Bhandari V. Pitfalls, problems, and progress in bronchopulmonary dysplasia. Pediatrics. 2009; 123:1562-73.
• [2]Tang JR, Karumanchi SA, Seedorf G, Markham N, Abman SH. Excess soluble vascular endothelial growth factor receptor-1 in amniotic fluid impairs lung growth in rats: linking preeclampsia with bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol. 2012; 302(1):L36-46.
• [3]Hogmalm A, Bäckström E, Bry M, Lappalainen U, Lukkarinen HP, Bry K. Role of CXC chemokine receptor-2 in a murine model of bronchopulmonary dysplasia. Am J Respir Cell Mol Biol. 2012; 47(6):746-58.
• [4]Zhu Y, Fu J, You K, Jin L, Wang M, Lu D et al.. Changes in pulmonary tissue structure and KL-6/MUC1 expression in a newborn rat model of hyperoxia-Induced bronchopulmonary dysplasia. Exp Lung Res. 2013; 9(10):417-26.
• [5]Dicken J, Mildner A, Leshkowitz D, Touw IP, Hantisteanu S, Jung S et al.. Transcriptional Reprogramming of CD11b(+)Esam(hi) Dendritic Cell Identity and Function by Loss of Runx3. PLoS One. 2013; 8(10): Article ID e77490
• [6]Homma N, Tamura G, Honda T, Matsumoto Y, Nishizuka S, Kawata S et al.. Spreading of methylation within RUNX3 CpG island in gastric cancer. Cancer Sci. 2006; 97(1):51-6.
• [7]Lee JM, Kwon HJ, Lai WF, Jung HS. Requirement of Runx3 in pulmonary vasculogenesis. Cell Tissue Res. 2014; 356(2):445-9.
• [8]Lee KS, Lee YS, Lee JM, Ito K, Cinghu S, Kim JH et al.. Runx3 is required for the differentiation of lung epithelial cells and suppression of lung cancer. Oncogene. 2010; 29(23):3349-61.
• [9]Ge MH, Chen C, Xu JJ, Ling ZQ. Critical regions and spreading of runt-related transcription factor-3 C-phosphate-G (CpG) island methylation in human salivary gland adenoid cystic carcinoma. Hum Pathol. 2011; 42(12):1862-72.
• [10]Deng T, Zhang Y. 5-Aza-2′-deoxycytidine reactivates expression of RUNX3 by deletion of DNA methyltransferases leading to caspase independent apoptosis in colorectal cancer Lovo cells. Biomed Pharmacother. 2009; 63(7):492-500.
• [11]Fujii S, Fukamachi K, Tsuda H, Ito K, Ito Y, Ochiai A. RAS oncogenic signal upregulates EZH2 in pancreatic cancer. Biochem Biophys Res Commun. 2012; 417:1074-9.
• [12]Agger K, Cloos PA, Christensen J, Pasini D, Rose S, Rappsilber J et al.. UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development. Nature. 2007; 449(7163):731-4.
• [13]Guo HB, Guo H. Mechanism of histone methylation catalyzed by protein lysine methyltransferase SET7/9 and origin of product specificity. Proc Natl Acad Sci U S A. 2007; 104:8797-802.
• [14]Fujii S, Ito K, Ito Y, Ochiai A. Enhancer of zeste homologue 2 (EZH2) down-regulates RUNX3 by increasing histone H3 methylation. J Biol Chem. 2008; 283:17324-32.
• [15]Denis H, Ndlovu MN, Fuks F. Regulation of mammalian DNA methyltransferases: a route to new mechanisms. EMBO Rep. 2011; 12:647-56.
• [16]Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell. 1999; 99:247-57.
• [17]Jung Y, Park J, Kim TY, Park JH, Jong HS, Im SA et al.. Potential advantages of DNA methyltransferase 1 (DNMT1)-targeted inhibition for cancer therapy. J Mol Med (Berl). 2007; 85(10):1137-48.
• [18]Parker RA, Lindstrom DP, Cotton RB. Evidence from twin study implies possible genetic susceptibility to bronchopulmonary dysplasia. Semin Perinatol. 1996; 20(3):206-9.
• [19]Bhandari V, Bizzarro MJ, Shetty A, Zhong X, Page GP, Zhang H et al.. Familial and genetic susceptibility to major neonatal morbidities in preterm twins. Pediatrics. 2006; 117(6):1901-6.
• [20]Cohen J, Van Marter LJ, Sun Y, Allred E, Leviton A, Kohane IS. Perturbation of gene expression of the chromatin remodeling pathway in premature newborns at risk for bronchopulmonary dysplasia. Genome Biol. 2007; 8:R210. BioMed Central Full Text
• [21]Londhe VA, Sundar IK, Lopez B, Maisonet TM, Yu Y, Aghai ZH et al.. Hyperoxia impairs alveolar formation and induces senescence through decreased histone deacetylase activity and up-regulation of p21 in neonatal mouse lung. Pediatr Res. 2011; 69(5 Pt 1):371-7.
• [22]Zhu L, Li H, Tang J, Zhu J, Zhang Y. Hyperoxia arrests alveolar development through suppression of histone deacetylases in neonatal rats. Pediatr Pulmonol. 2012; 47:264-74.
• [23]Ni W, Lin N, He H, Zhu J, Zhang Y. Lipopolysaccharide induces up-regulation of TGF-α through HDAC2 in a rat model of bronchopulmonary dysplasia. PLoS One. 2014; 9: Article ID e91083
• [24]Lee JJ, Murphy GF, Lian CG. Melanoma epigenetics: novel mechanisms, markers, and medicines. Lab Invest. 2014; 94:822-38.
• [25]You K, Xu X, Fu J, Xu S, Yue X, Yu Z et al.. Hyperoxia disrupts pulmonary epithelial barrier in newborn rats via the deterioration of occludin and ZO-1. Respir Res. 2012; 13:36. BioMed Central Full Text
• [26]Yang H, Fu J, Xue X, Yao L, Qiao L, Hou A, et al. Epithelial-mesenchymal transitions in bronchopulmonary dysplasia of newborn rats. Pediatr Pulmonol. 2014;11.
• [27]Yeh YY. Substrate utilization for phosphatidylcholine synthesis by type II pneumocytes of neonatal rats. Pediatr Res. 1991; 30:55-61.
• [28]Gruzieva O, Merid SK, Melén E. An update on epigenetics and childhood respiratory diseases. Paediatr Respir Rev. 2014; 15(4):348-54.
• [29]Jo P, Jung K, Grade M, Conradi LC, Wolff HA, Kitz J et al.. CpG island methylator phenotype infers a poor disease-free survival in locally advanced rectal cancer. Surgery. 2012; 151(4):564-70.
• [30]Yu GP, Ji Y, Chen GQ, Huang B, Shen K, Wu S et al.. Application of RUNX3 gene promoter methylation in the diagnosis of non-small cell lung cancer. Oncol Lett. 2012; 3:159-62.
• [31]Xiong L, Wang F, Huang X, Liu ZH, Zhao T, Wu LY et al.. DNA demethylation regulates the expression of miR-210 in neural progenitor cells subjected to hypoxia. FEBS J. 2012; 279:4318-26.
• [32]Dawson JA, Kamlin CO, Vento M, Wong C, Cole TJ, Donath SM et al.. Defining the reference range for oxygen saturation for infants after birth. Pediatrics. 2010; 125:e1340-7.
• [33]Vastenhouw NL, Schier AF. Bivalent histone modifications in early embryogenesis. Curr Opin Cell Biol. 2012; 24:374-86.
• [34]Bishop AE. Pulmonary epithelial stem cells. Cell Prolif. 2004; 37:89-96.
• [35]Lee JM, Shin JO, Cho KW, Hosoya A, Cho SW, Lee YS et al.. Runx3 is a crucial regulator of alveolar differentiation and lung tumorigenesis in mice. Differentiation. 2011; 81(4):261-8.
• [36]Viet CT, Dang D, Achdjian S, Ye Y, Katz SG, Schmidt BL. Decitabine rescues cisplatin resistance in head and neck squamous cell carcinoma. PLoS One. 2014; 9(11): Article ID e112880
• [37]Kawaguchi A, Ochi H, Sudou N, Ogino H. Comparative expression analysis of the H3K27 demethylases, JMJD3 and UTX, with the H3K27 methylase, EZH2, in Xenopus. Int J Dev Biol. 2012; 56(4):295-300.
• [38]Miranda TB, Cortez CC, Yoo CB, Liang G, Abe M, Kelly TK et al.. DZNep is a global histone methylation inhibitor that reactivates developmental genes Not silenced by DNA methylation. Mol Cancer Ther. 2009; 8:1579-88.
• [39]Kodach LL, Jacobs RJ, Heijmans J, van Noesel CJ, Langers AM, Verspaget HW et al.. The role of EZH2 and DNA methylation in the silencing of the tumour suppressor RUNX3 in colorectal cancer. Carcinogenesis. 2010; 31:1567-75.