Inhibitory Effect of MicroRNA-34a on Retinal Pigment Epithelial Cell Proliferation and Migration
Inhibitory Effect of MicroRNA-34a on Retinal Pigment Epithelial Cell Proliferation and Migration
复制标题
microRNA-34a对视网膜色素上皮细胞增殖和迁移的抑制作用。
DOI：
10.1167/iovs.13-11873
复制
发表时间：
2013-10-01
影响因子：
4.4
通讯作者：
Tu, LiLi
中科院分区：
文献类型：
--
作者：
Hou, Qiang;Tang, Jiang;Tu, LiLi
目的
视网膜色素上皮（RPE）细胞在诸如增殖性玻璃体视网膜病变、AMD和糖尿病视网膜病变的眼科疾病中起重要作用。MicroRNA-34 a（miR-34 a）在细胞增殖、迁移、分化和凋亡的调控中起重要作用。在本研究中，我们探讨了miR-34 a对RPE细胞的影响。
方法
实时荧光定量PCR检测ARPE-19细胞中miR-34 a的表达水平。将MicroRNA模拟物和小干扰RNA（siRNA）瞬时转染到RPE细胞中。WST-1增殖实验检测转染细胞的增殖能力，transwell实验和体外划痕实验检测转染细胞的迁移能力。Western blotting检测目的蛋白的表达或活化。
结果
与融合后细胞相比，MicroRNA-34 a在亚融合的ARPE-19细胞中显著下调。miR-34 a的导入抑制了RPE细胞的增殖和迁移能力，但没有明显的细胞凋亡。在miR-34 a转染的细胞中，许多重要的增殖和/或迁移相关分子如c-Met、CDK 2、CDK 4、CDK 6、E2 F1和磷酸化Cdc 2（p-Cdc 2）下调。针对c-Met设计的小干扰RNA也抑制RPE细胞的增殖和迁移，并下调CDK 2、CDK 6、E2 F1和p-Cdc 2。
结论
MicroRNA-34 a在亚融合的RPE细胞中下调。microRNA-34 a可通过下调其靶点c-Met等细胞周期相关分子来抑制RPE细胞的增殖和迁移。我们的结果表明miR-34 a参与了RPE细胞的调控。
PURPOSE
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.
METHODS
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.
RESULTS
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.
CONCLUSIONS
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.