McDonnell Fiona S, McNally Sara A, Clark Abbot F, O'Brien Colm J, Wallace Deborah M
*UCD School of Medicine and Medical Science, University College Dublin †Department of Ophthalmology, Mater Misericordiae University Hospital, Dublin, Ireland ‡Department of Cell Biology & Immunology and the North Texas Eye Research Institute, University of North Texas Health Science Center, Ft Worth, TX.
J Glaucoma. 2016 Oct;25(10):e834-e842. doi: 10.1097/IJG.0000000000000453.
Glaucoma is an optic neuropathy that affects 60 million people worldwide. There is an underlying fibrosis associated with the lamina cribrosa (LC) in glaucoma. DNA methylation is well established in regulating fibrosis and may be a therapeutic target for glaucoma. The purpose of this study was to compare global DNA methylation levels in primary human normal (NLC) and glaucomatous (GLC) cells, and to investigate DNA methylation in driving fibrosis through regulation of transforming growth factor β1 (TGFβ1).
LC cells were cultured from normal and glaucomatous human donors. Global methylation was assessed by ELISA. qPCR was conducted for DNA methyltransferases (DNMTs), methyl-CpG-binding protein 2 (MeCP2), TGFβ 1 and 2, collagen 1α1 (COL1A1), and α-smooth muscle actin (αSMA). TGFβ1 and DNMT1 were examined by immunofluorescence. Methylation of the TGFβ1 promoter was determined by methylation-specific PCR (MSP).
Global DNA methylation demonstrated an increase in GLC compared with NLC cells (P<0.05). The previously mentioned methylation and matrix genes were increased in GLC compared with NLC cells (P<0.05). Immunofluorescence showed increased TGFβ1 and DNMT1 in GLC compared with NLC cells. MSP showed increased unmethylated DNA in the TGFβ1 promoter of GLC compared with NLC cells.
We found increased expression of fibrotic genes in GLC cells and demonstrated an increase in global DNA methylation and in associated enzymes in GLC cells. Furthermore, we showed decreased promoter methylation of TGFβ1 in GLC cells. Determining a role for methylation in glaucoma and in regulating TGFβ1 may provide a novel therapeutic approach.
青光眼是一种影响全球6000万人的视神经病变。青光眼存在与筛板（LC）相关的潜在纤维化。DNA甲基化在调节纤维化过程中已得到充分证实，可能是青光眼的一个治疗靶点。本研究的目的是比较原代人正常（NLC）和青光眼（GLC）细胞中的整体DNA甲基化水平，并通过调节转化生长因子β1（TGFβ1）来研究DNA甲基化在驱动纤维化中的作用。
从正常和青光眼患者的供体中培养LC细胞。通过酶联免疫吸附测定（ELISA）评估整体甲基化。对DNA甲基转移酶（DNMTs）、甲基化CpG结合蛋白2（MeCP2）、TGFβ 1和2、胶原蛋白1α1（COL1A1）以及α平滑肌肌动蛋白（αSMA）进行定量聚合酶链反应（qPCR）。通过免疫荧光检测TGFβ1和DNMT1。通过甲基化特异性聚合酶链反应（MSP）测定TGFβ1启动子的甲基化。
与NLC细胞相比，GLC细胞的整体DNA甲基化增加（P<0.05）。与NLC细胞相比，GLC细胞中上述甲基化和基质基因增加（P<0.05）。免疫荧光显示，与NLC细胞相比，GLC细胞中TGFβ1和DNMT1增加。MSP显示，与NLC细胞相比，GLC细胞中TGFβ1启动子的未甲基化DNA增加。
我们发现GLC细胞中纤维化基因的表达增加，并证明GLC细胞中整体DNA甲基化及其相关酶增加。此外，我们还表明GLC细胞中TGFβ1启动子甲基化减少。确定甲基化在青光眼中的作用以及在调节TGFβ1中的作用可能提供一种新的治疗方法。
