抑制DNA甲基化和甲基CpG结合蛋白2可抑制视网膜色素上皮细胞转分化：与增殖性玻璃体视网膜病变的相关性
Inhibition of DNA Methylation and Methyl-CpG-Binding Protein 2 Suppresses RPE Transdifferentiation: Relevance to Proliferative Vitreoretinopathy.
作者信息
He Shikun, Barron Ernesto, Ishikawa Keijiro, Nazari Khanamiri Hossein, Spee Chris, Zhou Peng, Kase Satoru, Wang Zhuoshi, Dustin Laurie Diane, Hinton David R
机构信息
Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States 2Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, California, Unit.
Doheny Eye Institute, Los Angeles, California, United States.
出版信息
Invest Ophthalmol Vis Sci. 2015 Aug;56(9):5579-89. doi: 10.1167/iovs.14-16258.
PURPOSE
The purpose of this study was to evaluate expression of methyl-CpG-binding protein 2 (MeCP2) in epiretinal membranes from patients with proliferative vitreoretinopathy (PVR) and to investigate effects of inhibition of MeCP2 and DNA methylation on transforming growth factor (TGF)-β-induced retinal pigment epithelial (RPE) cell transdifferentiation.
METHODS
Expression of MeCP2 and its colocalization with cytokeratin and α-smooth muscle actin (α-SMA) in surgically excised PVR membranes was studied using immunohistochemistry. The effects of 5-AZA-2'-deoxycytidine (5-AZA-dC) on human RPE cell migration and viability were evaluated using a modified Boyden chamber assay and the colorimetric 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. Expression of RASAL1 mRNA and its promoter region methylation were evaluated by real-time PCR and methylation-specific PCR. Effects of 5-AZA-dC on expression of α-SMA, fibronectin (FN), and TGF-β receptor 2 (TGF-β R2) and Smad2/3 phosphorylation were analyzed by Western blotting. Effect of short interfering RNA (siRNA) knock-down of MeCP2 on expression of α-SMA and FN induced by TGFβ was determined.
RESULTS
MeCP2 was abundantly expressed in cells within PVR membranes where it was double labeled with cells positive for cytokeratin and α-SMA. 5-AZA-dC inhibited expression of MeCP2 and suppressed RASAL1 gene methylation while increasing expression of the RASAL1 gene. Treatment with 5-AZA-dC significantly suppressed the expression of α-SMA, FN, TGF-β R2 and phosphorylation of Smad2/3 and inhibited RPE cell migration. TGF-β induced expression of α-SMA, and FN was suppressed by knock-down of MeCP2.
CONCLUSIONS
MeCP2 and DNA methylation regulate RPE transdifferentiation and may be involved in the pathogenesis of PVR.
目的
本研究旨在评估甲基化CpG结合蛋白2（MeCP2）在增生性玻璃体视网膜病变（PVR）患者视网膜前膜中的表达，并探讨抑制MeCP2和DNA甲基化对转化生长因子（TGF）-β诱导的视网膜色素上皮（RPE）细胞转分化的影响。
方法
采用免疫组织化学方法研究手术切除的PVR膜中MeCP2的表达及其与细胞角蛋白和α-平滑肌肌动蛋白（α-SMA）的共定位。使用改良的Boyden小室试验和比色3-（4,5-二甲基噻唑-2）-2,5-二苯基四氮唑溴盐（MTT）试验评估5-氮杂-2'-脱氧胞苷（5-AZA-dC）对人RPE细胞迁移和活力的影响。通过实时PCR和甲基化特异性PCR评估RASAL1 mRNA的表达及其启动子区域甲基化。通过蛋白质免疫印迹分析5-AZA-dC对α-SMA、纤连蛋白（FN）和TGF-β受体2（TGF-β R2）表达以及Smad2/3磷酸化的影响。确定小干扰RNA（siRNA）敲低MeCP2对TGFβ诱导的α-SMA和FN表达的影响。
结果
MeCP2在PVR膜内的细胞中大量表达，且与细胞角蛋白和α-SMA阳性细胞呈双重标记。5-AZA-dC抑制MeCP2的表达，抑制RASAL1基因甲基化，同时增加RASAL1基因的表达。用5-AZA-dC处理可显著抑制α-SMA、FN、TGF-β R2的表达以及Smad2/3的磷酸化，并抑制RPE细胞迁移。敲低MeCP2可抑制TGF-β诱导的α-SMA和FN表达。
结论
MeCP2和DNA甲基化调节RPE转分化，可能参与PVR的发病机制。
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