The 4 th People's Hospital of Shenyang, Huanghe South Street No. 20, Huanggu District, 110031, Shenyang, Liaoning Province, China.
J Mol Histol. 2022 Jun;53(3):561-571. doi: 10.1007/s10735-022-10070-0. Epub 2022 Mar 23.
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.
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.
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.
Collectively, data in the present study indicated that targeting the miR-338-3p/SLC1A5 axis could block high glucose-induced ferroptosis in RPE cells.
氧化应激诱导的细胞铁死亡发生在糖尿病视网膜病变（DR）的发病机制中，但详细的分子机制尚不清楚。本研究旨在探讨这一问题。
体外采用高糖（30 mM）处理视网膜色素上皮（RPE）细胞，模拟体内 DR 进展的真实情况。通过 MTT 检测和台盼蓝染色检测细胞活力。通过 Real-Time qPCR 和 Western Blot 分析检测基因表达。FCM 用于检测细胞凋亡和 ROS 生成。双荧光素酶报告基因系统检测用于验证靶向部位。
高糖增加活性氧（ROS）水平，促进 RPE 细胞铁死亡，并抑制细胞增殖和活力，这一过程可被铁死亡抑制剂 ferrostatin-1 和 ROS 清除剂 N-乙酰-L-半胱氨酸（NAC）共同处理细胞所逆转。此外，我们筛选出一个在这个过程中起重要作用的 miR-338-3p/ASCT2（SLC1A5）轴。机制上，miR-338-3p 通过靶向 SLC1A5 的 3'非翻译区（3'UTR）来抑制和降解 SLC1A5，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 过表达载体转染入 RPE 细胞，随后进行的增益和失能实验验证了 miR-338-3p 消融和 SLC1A5 上调均可消除高糖对 RPE 细胞增殖、活力、铁死亡和 ROS 生成的调节作用。
综上所述，本研究数据表明，靶向 miR-338-3p/SLC1A5 轴可阻断高糖诱导的 RPE 细胞铁死亡。
