Background

Oxidative stress-induced cell ferroptosis occurs during the pathogenesis of diabetic retinopathy (DR), but the detailed molecular mechanisms are still unclear. The present study aimed to investigate this issue.

Materials and methods

The retinal pigment epithelium (RPE) was treated with high glucose (30 mM) in vitro to mimic the realistic conditions of DR progression in vivo. Cell viability was determined by MTT assay and trypan blue staining assay. Gene expressions were examined by Real-Time qPCR and Western Blot analysis. FCM was used to detect cell apoptosis and ROS generation. Dual-luciferase reporter gene system assay was used to verify the targeting sites.

Results

High glucose increased reactive oxygen species (ROS) levels, promoted cell ferroptosis, and suppressed cell proliferation and viability in RPE, which were reversed by co-treating cells with both a ferroptosis inhibitor ferrostatin-1 and an ROS scavenger, N-acetyl-L-Cysteine (NAC). In addition, we screened out a miR-338-3p/ASCT2 (SLC1A5) axis that played an important role in this process. Mechanistically, miR-338-3p targeted the 3’ untranslated regions (3’UTR) of SLC1A5 for its inhibition and degradation, and high glucose downregulated SLC1A5 by upregulating miR-338-3p in RPE cells. Next, the miR-338-3p inhibitor and SLC1A5 overexpression vectors were delivered into the RPE cells, and the following gain- and loss-of-function experiments validated that both miR-338-3p ablation and SLC1A5 upregulation abrogated the regulating effects of high glucose on cell proliferation, viability, ferroptosis and ROS production in RPE cells.

Conclusions

Collectively, data in the present study indicated that targeting the miR-338-3p/SLC1A5 axis could block high glucose-induced ferroptosis in RPE cells.

中文翻译：

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一种新的 miR-338-3p/SLC1A5 轴重编程视网膜色素上皮细胞以增加其对高糖诱导的细胞铁死亡的抵抗力

背景

氧化应激诱导的细胞铁死亡发生在糖尿病视网膜病变（DR）的发病过程中，但其详细的分子机制仍不清楚。本研究旨在调查这个问题。

材料和方法

视网膜色素上皮 (RPE)在体外用高葡萄糖 (30 mM) 处理，以模拟体内DR 进展的现实条件。通过MTT测定和台盼蓝染色测定确定细胞活力。通过实时 qPCR 和蛋白质印迹分析检查基因表达。FCM用于检测细胞凋亡和ROS产生。双荧光素酶报告基因系统测定用于验证靶向位点。

结果

高葡萄糖会增加活性氧 (ROS) 水平，促进细胞铁死亡，并抑制 RPE 中的细胞增殖和活力，这通过用铁死亡抑制剂 ferrostatin-1 和 ROS 清除剂 N-乙酰-L 共同处理细胞来逆转-半胱氨酸（NAC）。此外，我们筛选出在该过程中发挥重要作用的 miR-338-3p/ASCT2 (SLC1A5) 轴。机制上，miR-338-3p 靶向 SLC1A5 的 3' 非翻译区 (3'UTR) 抑制和降解，高糖通过上调 RPE 细胞中的 miR-338-3p 来下调 SLC1A5。接下来，将 miR-338-3p 抑制剂和 SLC1A5 过表达载体递送到 RPE 细胞中，

结论

总的来说，本研究中的数据表明，靶向 miR-338-3p/SLC1A5 轴可以阻断 RPE 细胞中高糖诱导的铁死亡。