Inhibition of DNA Methylation and Methyl-CpG-Binding Protein 2 Suppresses RPE Transdifferentiation: Relevance to Proliferative Vitreoretinopathy
Inhibition of DNA Methylation and Methyl-CpG-Binding Protein 2 Suppresses RPE Transdifferentiation: Relevance to Proliferative Vitreoretinopathy
复制标题
DOI：
10.1167/iovs.14-16258
复制
发表时间：
2015-08-01
影响因子：
4.4
通讯作者：
Hinton, David R.
中科院分区：
文献类型：
--
作者：
He, Shikun;Barron, Ernesto;Hinton, David R.
目的.本研究的目的是评估增生性玻璃体视网膜病变（PVR）患者视网膜前膜中甲基CpG结合蛋白2（MeCP 2）的表达，并探讨抑制MeCP 2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了手术切除的PVR膜中MeCP 2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。用改良的Boyden小室法和3-（4，5-二甲基噻唑基-2）-2，5-二苯基四氮唑盐（MTT）比色法观察5-AZA-2 '-脱氧胞苷（5-AZA-dC）对人RPE细胞迁移和活力的影响。采用实时荧光定量PCR和甲基化特异性PCR检测RASAL 1 mRNA的表达及其启动子区甲基化。通过蛋白质印迹分析57 AZA-dC对α-SMA、纤连蛋白（EN）和TGF-β受体2（TGF-β R2）的表达以及Smad 2/3磷酸化的影响。测定MeCP 2的短干扰RNA（siRNA）敲低对TGF β诱导的α-SMA和FN表达的影响。MeCP 2在PVR膜内的细胞中大量表达，其中它被细胞角蛋白和α-SMA阳性的细胞双重标记。5-AZA-dC抑制MeCP 2的表达，抑制RASAL 1基因甲基化，同时增加RASA 1基因的表达。5-AZA-dC处理显著抑制了α-SMA、EN、TGF-β R2的表达和Smad 2/3的磷酸化，并抑制了RPE细胞的迁移。TGF-β诱导。MeCP 2的敲低可抑制α-SMA和FN的表达。MeCP 2和DNA甲基化调节RPE转分化，并可能参与PVR的发病机制。
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)-beta-induced retinal pigment epithelial (RPE) cell transdilfferentiation.METHODS. Expression of MeCP2 and its colocalization with cytokeratin and alpha-smooth muscle actin (alpha-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-diphenyltetrazolitun bromide (MTT) assay. Expression of RASAL1 mRNA and its promoter region methylation were evaluated by real-tune PCR and methylation-specific PCR. Effects of 57AZA-dC on expression of a-SMA, fibronectin (EN), and TGF-beta receptor 2 (TGF-beta R2) and Smad2/3 phosphorylation were analyzed by Western blotting. Effect of short interfering RNA (siRNA) knock-down of MeCP2 on expression of alpha-SMA and FN induced by TGF beta was determined.RESULTS. MeCP2 was abundantly expressed in cells within PVR membranes where it was double labeled with cells positive for cytokeratin and alpha-SMA. 5-AZA-dC inhibited expression of MeCP2 and suppressed RASAL1 gene methylation while increasing expression of the RASA1 gene. Treatment with 5-AZA-dC significantly suppressed the expression of alpha-SMA, EN, TGF-beta R2 and phosphorlation of Smad2/3 and inhibited RPE cell migration. TGF-beta induced. expression of alpha-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.