Li Hongyang, Gong Yan, Qian Haiyan, Chen Tingjun, Liu Zihao, Jiang Zhaocai, Wei Shihui
Department of Opthalmology, The Chinese People's Liberation Army General Hospital, Beijing 100000, P.R. China.
Mol Med Rep. 2015 Aug;12(2):2793-9. doi: 10.3892/mmr.2015.3736. Epub 2015 May 6.
Light-induced retinal injury is clinically and experimentally well-documented. It may be categorized into three types: Photothermal, photomechanical and photochemical injuries. To date, the variation in the hsa-miR-183/96/182 cluster and its potential target genes in human primary retinal pigment epithelial (RPE) cells, following visible light exposure, has not been reported. In the present study, RPE cells were exposed to 4 h of constant visible light. The expression of the hsa-miR-183/96/182 cluster was determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and its potential target genes were investigated. Additionally, hsa-miR-183, hsa-miR-96, hsa-miR-182 and has-miR-183/96/182 mimics were designed and synthesized in vitro, and transfected into the RPE cells. Subsequently, the expression of brain-derived neurotrophic factor (BDNF) mRNA and protein was measured, using RT-qPCR and western blotting, respectively. The regulation of miRNAs to the BDNF gene were then validated using a dual luciferase reporter gene assay system. The expression of hsa-miR-183, hsa-miR-96 and hsa-miR-182 significantly increased in RPE cells following 4 h of visible light exposure, compared with RPE cells that had been exposed to dark conditions (P<0.01). Following RPE cell transfection with mimics, BDNF mRNA and protein expression in the RPE cells was significantly downregulated compared with control RPE cells (P<0.05, P<0.01, respectively). Similarly, the ratio of Renilla luciferase/firefly luciferase significantly decreased in the RPE cells of the mimic + wild type (WT) group compared with cells of the psiCHECK(TM)-2 (a vector lacking the sequence of the BDNF gene), wild type and mimic + mutation groups (P<0.05, P<0.01). The present study suggests that BDNF is a target gene of the has-miR-183-96-182 cluster in RPE cells. The present study suggests an underlying protective mechanism against retinal light injury and may provide a novel target for the prevention and treatment of light-induced retinal injury.
光诱导的视网膜损伤在临床和实验上都有充分的文献记载。它可分为三种类型：光热损伤、光机械损伤和光化学损伤。迄今为止，尚未有关于人原代视网膜色素上皮（RPE）细胞在可见光照射后hsa-miR-183/96/182簇及其潜在靶基因变化的报道。在本研究中，将RPE细胞暴露于4小时的持续可见光下。使用逆转录定量聚合酶链反应（RT-qPCR）测定hsa-miR-183/96/182簇的表达，并研究其潜在靶基因。此外，体外设计并合成了hsa-miR-183、hsa-miR-96、hsa-miR-182和has-miR-183/96/182模拟物，并将其转染到RPE细胞中。随后，分别使用RT-qPCR和蛋白质印迹法测量脑源性神经营养因子（BDNF）mRNA和蛋白质的表达。然后使用双荧光素酶报告基因检测系统验证miRNA对BDNF基因的调控。与暴露于黑暗条件下的RPE细胞相比，RPE细胞在可见光照射4小时后，hsa-miR-183、hsa-miR-96和hsa-miR-182的表达显著增加（P<0.01）。用模拟物转染RPE细胞后，与对照RPE细胞相比，RPE细胞中BDNF mRNA和蛋白质表达显著下调（分别为P<0.05，P<0.01）。同样，与psiCHECK(TM)-2（一种缺乏BDNF基因序列的载体）、野生型和模拟物+突变组的细胞相比，模拟物+野生型（WT）组的RPE细胞中海肾荧光素酶/萤火虫荧光素酶的比率显著降低（P<0.05，P<0.01）。本研究表明BDNF是RPE细胞中has-miR-183-96-182簇的靶基因。本研究提示了一种针对视网膜光损伤的潜在保护机制，并可能为光诱导的视网膜损伤的预防和治疗提供新的靶点。
