BACKGROUND Mitochondrial function in retinal pigmented epithelial (RPE) cells and extracellular vesicle (EV) formation/release are related through the lysosomal and exocytotic pathways that process and eliminate intracellular material, including mitochondrial fragments. We propose that RPE cells with impaired mitochondria will release EVs containing mitochondrial miRNAs that reflect the diminished capacity of mitochondria within these cells. METHODS We screened ARPE-19 cells for miRNAs that localize to the mitochondria, exhibit biological activity, and are present in EVs released by both untreated cells and cells treated with rotenone to induce mitochondrial injury. EVs were characterized by vesicle size, size distribution, presence of EV biomarkers: CD81, CD63, and syntenin-1, miRNA cargo, and number concentration of EVs released per cell. RESULTS We found that miR-494-3p was enriched in ARPE-19 mitochondria. Knockdown of miR-494-3p in ARPE-19 cells decreased ATP production and mitochondrial membrane potential in a dose-dependent manner, and decreased basal oxygen consumption rate and maximal respiratory capacity. Increased number of EVs released per cell and elevated levels of miR-494-3p in EVs released from ARPE-19 cells treated with rotenone were also measured. CONCLUSIONS ARPE-19 mitochondrial function is regulated by miR-494-3p. Elevated levels of miR-494-3p in EVs released by ARPE-19 cells indicate diminished capacity of the mitochondria within these cells. GENERAL SIGNIFICANCE EV miR-494-3p is a potential biomarker for RPE mitochondrial dysfunction, which plays a central role in non-neovascular age-related macular degeneration, and may be a diagnostic biomarker for monitoring the spread of degeneration to neighboring RPE cells in the retina.

中文翻译：

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线粒体受损的ARPE-19细胞释放细胞外小泡miR-494-3p。

背景技术视网膜色素上皮（RPE）细胞中的线粒体功能和细胞外囊泡（EV）的形成/释放是通过溶酶体和胞吐途径进行的，这些途径加工并消除了细胞内物质，包括线粒体片段。我们建议线粒体受损的RPE细胞将释放包含线粒体miRNA的EV，反映这些细胞内线粒体的能力降低。方法我们从ARPE-19细胞中筛选了定位于线粒体，具有生物学活性并且存在于未处理细胞和鱼藤酮处理过的细胞中的线粒体，诱导线粒体损伤的miRNA。EV的特征是囊泡大小，大小分布，EV生物标志物（CD81，CD63和Syntenin-1）的存在，miRNA货物以及每个细胞释放的EV的数量浓度。结果我们发现miR-494-3p富含ARPE-19线粒体。敲低ARPE-19细胞中miR-494-3p的剂量依赖性降低了ATP的产生和线粒体膜电位，并降低了基础耗氧率和最大呼吸能力。还测量了由鱼藤酮处理过的ARPE-19细胞释放的每个细胞释放的EV数量增加和miR-494-3p水平升高。结论ARPE-19线粒体功能受miR-494-3p调控。ARPE-19细胞释放的EV中miR-494-3p的水平升高表明这些细胞内线粒体的能力降低。一般意义EV miR-494-3p是RPE线粒体功能障碍的潜在生物标志物，在非血管性年龄相关性黄斑变性中起着核心作用，