June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Genomewide DNA methylation analysis of human trabecular meshwork cells with dexamethasone stimulation
Author Affiliations & Notes
  • Akira Matsuda
    Department of Ophthalmology, Juntendo Univ School of Med, Tokyo, Japan
  • Nobuyuki Ebihara
    Department of Ophthalmology, Juntendo Univ School of Med, Tokyo, Japan
  • Akira Murakami
    Department of Ophthalmology, Juntendo Univ School of Med, Tokyo, Japan
  • Footnotes
    Commercial Relationships Akira Matsuda, None; Nobuyuki Ebihara, None; Akira Murakami, SEED(Japan) JP4855782 (P), SEED(Japan) JP5132958 (P)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 4003. doi:
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      Akira Matsuda, Nobuyuki Ebihara, Akira Murakami; Genomewide DNA methylation analysis of human trabecular meshwork cells with dexamethasone stimulation. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4003.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To investigate the change of DNA methylation status during glucocorticoid exposure using cultured human trabecular meshwork cells.

Methods: Human trabecular meshwork (TM) cells established from glaucoma patients (GTM) and normal control (NTM) were cultured with and without 100nM dexamethasone (DEX) for 14 days. Genome wide methylation analysis was carried out using Illumina 450K methylation analysis. Gene expression analysis was also carried out with Agilent 44K whole human genome array using cultured TM cells. The effect of DNA methylation in response to DEX treatments for gene expression was verified by DNA methyltransferase inhibitor (5-aza-2’-deoxycytidine) treatment and subsequent realtime PCR analysis.

Results: We found 174/297 cytosine-phosphate-guanine (CpG) sites showing significant demethylation and 102/184 CpG sites showing significant methylation by 2 week-DEX treatment in GTM/NTM, respectively. We found 23 sets of demethylated-CpG sites after DEX exposure with significant changes of corresponding gene expression, and 15 sets methylated-CpG sites after DEX exposure with significant changes of corresponding gene expression. Among them we further evaluated the expression of FKBP5, ZBTB16 and DUSP1. ZBTB16 gene-body methylation was observed after DEX treatment, accompanied with increased gene expression. 5-aza-2’-deoxycytidine-treatment of TM cells abolished the increase of ZBTB16 mRNA expression. DNA-demethylation of FKBP5 promoter region was observed with increased FKBP5 gene expression by DEX treatment. Promoter region of DUSP1 gene region is highly methylated in NTM cells. Significant DNA-demethylation and increase of DUSP1 gene expression was observed after DEX treatment in NTM cells but not in GTM cells.

Conclusions: DEX treatments induce the alteration of DNA methylation status in human TM cells. Epigenetic modification could affect TM gene expression profile.

Keywords: 533 gene/expression • 735 trabecular meshwork • 536 gene modifiers  
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