Zhou Yang, Yusufu Meilibanu, Zhang Ting, Wang Jing
Department of Ophthalmology, The Fifth Affiliated Hospital of Xinjiang Medical University, Henan Road No. 118, Ürümqi, 830011, Xinjiang, China.
Department of Eye Center, Qingdao Municipal Hospital (Group), Jiaozhou Road No.1, Qingdao, 266011, Shandong, China.
Cytotechnology. 2020 Oct 29;72(6):873-84. doi: 10.1007/s10616-020-00431-6.
Oxidative damages contributes to age-related macular degeneration (AMD) caused vision blindness, but the molecular mechanisms are still largely unknown.
This study managed to investigate this issue by conducting in vitro experiments.
Oxidative stress were evaluated by L-012 dye, DHE staining and MDA assay. CCK-8 and colony formation assay were conducted to examine cell proliferation. Cell death was evaluated by trypan blue staining and Annexin V-FITC/PI double staining method through flow cytometry (FCM). The binding sites of miR-23a and GLS1 mRNA were predicted by online miRDB database and validated by dual-luciferase reporter gene system. Real-Time qPCR for miR-23a levels and Western Blot for protein expressions.
The retinal pigment epithelial (RPE) cells (ARPE-19) were subjected to hydrogen peroxide (HO) stimulation to simulate AMD progression in vitro, and we identified a novel miR-23a/glutaminase-1 (GLS1) pathway that regulated HO induced oxidative damages in ARPE-19 cells. Mechanistically, HO induced oxidative stress, inhibited cell proliferation and induced cell death in ARPE-19 cells in a dose- and time-dependent manner. Also, HO stimulation hindered cell invasion, migration and glutamine uptake in ARPE-19 cells. Interestingly, we proved that HO increased miR-23a levels, while downregulated glutaminase-1 (GLS1) in ARPE-19 cells, and miR-23a targeted 3' untranslated region (3'UTR) of GLS1 mRNA for GLS1 degradation. Finally, our data suggested that silencing miR-23a upregulated GLS1 to reverse the detrimental effects of HO treatment on ARPE-19 cells.
In general, analysis of the data suggested that miR-23a ablation upregulated GLS1 to attenuate HO stimulation induced oxidative damages in ARPE-19 cells in vitro, and this study broadened our knowledge in this field, which might help to provide novel theranostic signatures for AMD.
氧化损伤导致年龄相关性黄斑变性（AMD）引发视力丧失，但其分子机制仍 largely 未知。
本研究通过进行体外实验来探究此问题。
通过 L - 012 染料、DHE 染色和 MDA 测定评估氧化应激。进行 CCK - 8 和集落形成实验检测细胞增殖。通过台盼蓝染色和 Annexin V - FITC/PI 双染法经流式细胞术（FCM）评估细胞死亡。通过在线 miRDB 数据库预测 miR - 23a 与 GLS1 mRNA 的结合位点，并经双荧光素酶报告基因系统验证。采用实时定量 PCR 检测 miR - 23a 水平，采用蛋白质印迹法检测蛋白质表达。
视网膜色素上皮（RPE）细胞（ARPE - 19）接受过氧化氢（HO）刺激以在体外模拟 AMD 进展，我们鉴定出一条新的 miR - 23a/谷氨酰胺酶 - 1（GLS1）途径，该途径调节 HO 在 ARPE - 19 细胞中诱导的氧化损伤。机制上，HO 以剂量和时间依赖性方式在 ARPE - 19 细胞中诱导氧化应激、抑制细胞增殖并诱导细胞死亡。此外，HO 刺激阻碍 ARPE - 19 细胞的侵袭、迁移和谷氨酰胺摄取。有趣的是，我们证明 HO 增加 ARPE - 19 细胞中 miR - 23a 水平，同时下调谷氨酰胺酶 - 1（GLS1），且 miR - 23a 靶向 GLS1 mRNA 的 3'非翻译区（3'UTR）以降解 GLS1。最后，我们的数据表明沉默 miR - 23a 上调 GLS1 以逆转 HO 处理对 ARPE - 19 细胞的有害影响。
总体而言，数据分析表明 miR - 23a 缺失上调 GLS1 以减轻 HO 刺激在体外诱导的 ARPE - 19 细胞氧化损伤，本研究拓宽了我们在该领域的知识，这可能有助于为 AMD 提供新的诊疗标志物。
