School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China.
Invest Ophthalmol Vis Sci. 2013 Oct 3;54(10):6481-8. doi: 10.1167/iovs.13-11873.
Retinal pigment epithelial (RPE) cells play important roles in ophthalmologic diseases such as proliferative vitreoretinopathy, AMD, and diabetic retinopathy. MicroRNA-34a (miR-34a) has been reported to be important in the regulation of cell proliferation, migration, differentiation, and apoptosis. In this study, we explored the effects of miR-34a on RPE cells.
The expression level of miR-34a in subconfluent and postconfluent ARPE-19 cells was investigated with quantitative real-time PCR. MicroRNA mimic and small interfering RNA (siRNA) were transiently transfected into RPE cells. Transfected RPE cells were analyzed with WST-1 proliferation assay, and their migration was analyzed with transwell assay and in vitro scratch study. The expression or activation of target proteins was detected by Western blotting.
MicroRNA-34a was significantly downregulated in subconfluent ARPE-19 cells compared with postconfluent cells. Introduction of miR-34a inhibited the proliferation and migratory ability of RPE cells without obvious cell apoptosis. In miR-34a transfected cells, many important proliferation and/or migration related molecules such as c-Met, CDK2, CDK4, CDK6, E2F1, and phosphorylated-Cdc2 (p-Cdc2) were downregulated. Small interfering RNA designed to target c-Met also inhibited the proliferation and migration of RPE cells and downregulated CDK2, CDK6, E2F1, and p-Cdc2.
MicroRNA-34a is downregulated in subconfluent RPE cells. MicroRNA-34a can inhibit the proliferation and migration of RPE cells through downregulation of its targets c-Met and other cell cycle-related molecules. Our results indicated that miR-34a is involved in the regulation of RPE cells.
视网膜色素上皮（RPE）细胞在眼科疾病中发挥着重要作用，如增生性玻璃体视网膜病变、AMD 和糖尿病性视网膜病变。microRNA-34a（miR-34a）已被报道在细胞增殖、迁移、分化和凋亡的调控中具有重要作用。在本研究中，我们探讨了 miR-34a 对 RPE 细胞的影响。
用实时定量 PCR 检测亚汇合和汇合后 ARPE-19 细胞中 miR-34a 的表达水平。瞬时转染 microRNA 模拟物和小干扰 RNA（siRNA）至 RPE 细胞。用 WST-1 增殖测定分析转染的 RPE 细胞，用 Transwell 测定和体外划痕实验分析其迁移能力。用 Western 印迹检测靶蛋白的表达或激活。
与汇合细胞相比，亚汇合 ARPE-19 细胞中 miR-34a 的表达显著下调。引入 miR-34a 抑制了 RPE 细胞的增殖和迁移能力，而无明显的细胞凋亡。在转染 miR-34a 的细胞中，许多重要的增殖和/或迁移相关分子，如 c-Met、CDK2、CDK4、CDK6、E2F1 和磷酸化-Cdc2（p-Cdc2）被下调。靶向 c-Met 的小干扰 RNA 也抑制了 RPE 细胞的增殖和迁移，并下调了 CDK2、CDK6、E2F1 和 p-Cdc2。
miR-34a 在亚汇合的 RPE 细胞中下调。miR-34a 可通过下调其靶标 c-Met 和其他细胞周期相关分子来抑制 RPE 细胞的增殖和迁移。我们的研究结果表明，miR-34a 参与了 RPE 细胞的调控。
