Background

Oxidative damages contributes to age-related macular degeneration (AMD) caused vision blindness, but the molecular mechanisms are still largely unknown.

Objectives

This study managed to investigate this issue by conducting in vitro experiments.

Methods

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.

Results

The retinal pigment epithelial (RPE) cells (ARPE-19) were subjected to hydrogen peroxide (H₂O₂) stimulation to simulate AMD progression in vitro, and we identified a novel miR-23a/glutaminase-1 (GLS1) pathway that regulated H₂O₂ induced oxidative damages in ARPE-19 cells. Mechanistically, H₂O₂ induced oxidative stress, inhibited cell proliferation and induced cell death in ARPE-19 cells in a dose- and time-dependent manner. Also, H₂O₂ stimulation hindered cell invasion, migration and glutamine uptake in ARPE-19 cells. Interestingly, we proved that H₂O₂ 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 H₂O₂ treatment on ARPE-19 cells.

Conclusions

In general, analysis of the data suggested that miR-23a ablation upregulated GLS1 to attenuate H₂O₂ 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.

中文翻译：

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沉默 miR-23a 通过上调 GLS1 减弱过氧化氢 (H 2 O 2 ) 诱导的 ARPE-19 细胞氧化损伤：一项体外研究

 背景

氧化损伤会导致年龄相关性黄斑变性（AMD）导致视力失明，但其分子机制仍不清楚。

 目标

这项研究通过进行体外实验来研究这个问题。

 方法

通过 L-012 染料、DHE 染色和 MDA 测定评估氧化应激。进行CCK-8和集落形成测定来检查细胞增殖。通过流式细胞仪（FCM）台盼蓝染色和膜联蛋白V-FITC/PI双染色法评估细胞死亡。通过在线miRDB数据库预测miR-23a和GLS1 mRNA的结合位点，并通过双荧光素酶报告基因系统进行验证。实时 qPCR 检测 miR-23a 水平，蛋白质印迹检测蛋白质表达。

 结果

对视网膜色素上皮 (RPE) 细胞 (ARPE-19) 进行过氧化氢 (H ₂ O ₂ ) 刺激以在体外模拟 AMD 进展，我们发现了一条新的miR-23a /谷氨酰胺酶-1 ( GLS1 ) 通路来调节H ₂ O ₂诱导 ARPE-19 细胞氧化损伤。从机制上讲，H ₂ O ₂以剂量和时间依赖性方式诱导 ARPE-19 细胞氧化应激、抑制细胞增殖并诱导细胞死亡。此外，H ₂ O ₂刺激阻碍了 ARPE-19 细胞的细胞侵袭、迁移和谷氨酰胺摄取。有趣的是，我们证明H ₂ O ₂增加了ARPE-19细胞中的miR-23a水平，同时下调了谷氨酰胺酶-1 ( GLS1 )，并且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 H ₂ O ₂处理对 ARPE-19 细胞的有害影响。

 结论

总的来说，数据分析表明miR-23a消融上调GLS1以减弱H ₂ O ₂刺激诱导的ARPE-19细胞体外氧化损伤，这项研究拓宽了我们在这一领域的知识，这可能有助于提供新的治疗诊断方法。 AMD 的签名。