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Survival Improvement in Human Retinal Pigment Epithelial Cells via Fas Receptor Targeting by miR‐374a

机译:MIR-374A靶向人体视网膜色素上皮细胞的生存改善

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ABSTRACT Oxidative conditions of the eye could contribute to retinal cells loss through activating the Fas‐L/Fas pathway. This phenomenon is one of the leading causes of some ocular diseases like age‐related macular degeneration (AMD). By targeting proteins at their mRNA level, microRNAs (miRNAs) can regulate gene expression and cell function. The aim of the present study is to investigate Fas targeting by miR‐374a and find whether it can inhibit Fas‐mediated apoptosis in primary human retinal pigment epithelial (RPE) cells under oxidative stress. So, the primary human RPE cells were transfected with pre‐miR‐374a pLEX construct using polymeric carrier and were exposed to H 2 O 2 (200?μM) as an oxidant agent for induction of Fas expression. Fas expression at mRNA and protein level was evaluated by quantitative real‐time PCR and Western blot analysis, respectively. These results revealed that miR‐374a could prevent Fas upregulation under oxidative conditions. Moreover, Luciferase activity assay confirmed that Fas could be a direct target of miR‐374a. The cell viability studies demonstrated that caspase‐3 activity was negligible in miR‐374a treated cells compared to the controls. Our data suggest miR‐374a is a negative regulator of Fas death receptor which is able to enhance the cell survival and protect RPE cells against oxidative conditions. J. Cell. Biochem. 118: 4854–4861, 2017. ? 2017 Wiley Periodicals, Inc.
机译:摘要眼睛的氧化条件可以通过激活FAS-L / FAS途径有助于视网膜细胞损失。这种现象是某些眼部疾病的主要原因之一,如年龄相关的黄斑变性(AMD)。通过将蛋白质靶向它们的mRNA水平,MicroRNAs(miRNA)可以调节基因表达和细胞功能。本研究的目的是探讨MIR-374A的FAS靶向,并在氧化应激下抑制原发性人视网膜颜料上皮(RPE)细胞中的FAS介导的细胞凋亡。因此,使用聚合物载体用前miR-374a plex构建体转染原发性人RPE细胞,并将其暴露于H 2 O 2(200μm)作为氧化剂,用于诱导Fas表达。通过定量实时PCR和Western印迹分析评估mRNA和蛋白质水平的Fas表达。这些结果表明,miR-374a可以在氧化条件下预防FAS上调。此外,荧光素酶活性测定证实Fas可以是miR-374a的直接靶标。细胞活力研究表明,与对照相比,MiR-374A处理细胞中的Caspase-3活性可忽略不计。我们的数据建议MiR-374a是Fas死亡受体的负调节剂,其能够增强细胞存活率并保护RPE细胞免受氧化条件。 J.Cell。生物学习。 118:4854-4861,2017。 2017年Wiley期刊,Inc。

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