Abstract：
Exosomes are 40-100 nm nano-sized extracellular vesicles and are receiving increasing attention as novel structures that participate in intracellular communication. We previously found that miRNA-1 (miR-1) functions as a tumor suppressor in renal cell carcinoma (RCC). In this study, we investigated the function of exosomal miR-1 and the possibility that the exosome constitutes a tumor maker in RCC. First, we established the method to collect exosomes from cell lysates and human serum by a spin column-based method. Next, we assessed exosomes using Nanosight nanoparticle tracking analysis and Western blot analysis with exosome marker CD63. We confirmed that exosomes labeled with PKH26 fused with recipient cells. Moreover, miR-1 expression was elevated in RCC cells treated with exosomes derived from miR-1-transfected cells. Functional analyses showed that exosomal miR-1 significantly inhibited cell proliferation, migration and invasion compared to control treatment. Our analyses with TCGA database of RCCs showed that miR-1 expression was significantly downregulated in clinical RCC samples compared to that in normal kidney samples, and patients with low miR-1 expression had poorer overall survival in comparison to patients with high expression. Furthermore, RNA sequence analyses showed that expression levels of several genes were altered by exposure to exosomal miR-1. The analyses with TCGA database indicated that high expression of MYO15A was associated with a poorer outcome in RCC. In addition, RT-qPCR analysis of exosomes from clinical patients\' sera showed that MYO15A was significantly upregulated in RCC patients compared to that in healthy controls. This study showed that treatment with exosomal miR-1 might be an effective approach to treating RCCs. In addition, exosomal MYO15A could be a diagnostic tumor marker in RCCs.
摘要：
外泌体是40-100nm纳米大小的细胞外囊泡，作为参与细胞内通讯的新型结构越来越受到关注。我们先前发现miRNA-1（miR-1）在肾细胞癌（RCC）中起肿瘤抑制因子的作用。在这项研究中,我们研究了外泌体miR-1的功能以及外泌体构成RCC肿瘤标志物的可能性。首先,我们建立了通过基于旋转柱的方法从细胞裂解物和人血清中收集外泌体的方法。接下来,我们使用Nanosight纳米颗粒追踪分析和使用外泌体标志物CD63的Western印迹分析评估了外泌体.我们证实用PKH26标记的外泌体与受体细胞融合。此外，miR-1表达在用源自miR-1转染细胞的外泌体处理的RCC细胞中升高。功能分析显示，外泌体miR-1显著抑制细胞增殖，与对照处理相比，迁移和入侵。我们使用TCGARCC数据库的分析显示，与正常肾脏样本相比，临床RCC样本中miR-1表达显著下调。与miR-1高表达患者相比,miR-1低表达患者的总生存期较差.此外，RNA序列分析表明，暴露于外泌体miR-1会改变几种基因的表达水平。对TCGA数据库的分析表明，MYO15A的高表达与RCC的较差预后相关。此外,临床患者血清外泌体的RT-qPCR分析显示，与健康对照相比，RCC患者的MYO15A显著上调。这项研究表明，外泌体miR-1治疗可能是治疗RCC的有效方法。此外,外泌体MYO15A可能是RCC的诊断肿瘤标志物。
