Paracrine signaling between tumor and surrounding stromal cells is critical for the maintenance of tumor microenvironment during ovarian cancer progression. Small extracellular vesicles (sEVs; exosomes in particular) are nano-sized vesicles secreted actively by many cells including tumor cells and are found to have fundamental roles in intercellular communication through shuttling functional RNAs. Although microRNAs (also called miRNAs or miRs), small non-coding RNAs regulating gene expression, are selectively accumulated in tumor sEVs and can mediate intercellular communication, the exact biological mechanisms underlying the functions of exosomal miRNAs in ovarian tumor angiogenesis remain unclear. In this study, sEVs were isolated from conditioned medium of the human ovarian carcinoma cell line SKOV-3 using ExoQuick Exosome Precipitation Solution, and characterized by scanning electron microscopy, dynamic light scattering, and immunoblotting. To elucidate the possible paracrine effects on ovarian tumor cell-derived sEVs (TD-sEVs), we investigated the angiogenesis-related signaling events triggered by TD-sEVs in endothelial cells. Due to the possible role in ovarian tumor pathogenesis, we focused on miR-141-3p which was detected to be enriched in TD-sEVs compared with their corresponding donor cells. We identified that sEV transfer of miR-141-3p considerably reduced the expression levels of cytokine-inducible suppressors of cytokine signaling (SOCS)-5 leading to up-regulated JAK-STAT3 pathway in endothelial cells. We also observed that sEV-shuttled miR-141-3p may up-regulate the expression of VEGFR-2 in endothelial cells which leads to promoting endothelial cell migration and angiogenesis. The putative role of miR-141-3p shuttled by TD-sEVs in regulating VEGFR-2 expression was demonstrated by the ability of anti-miR-141-3p to rescue the promoting effects of TD-sEVs on the expression of VEGFR-2 in endothelial cells. Our results also revealed that TD-sEVs trigger the intracellular reactive oxygen species (ROS)-dependent activation of NF-κB signaling in endothelial cells. Taken together, our findings propose a novel model in which sEV transfer of epithelial ovarian cancer-secreted miR-141-3p plays as a significant mediator of intercellular communication, promoting endothelial cell angiogenesis.

中文翻译：

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来自上皮性卵巢癌细胞的含有 microRNA-141-3p 的小细胞外囊泡通过激活 JAK/STAT3 和 NF-κB 信号通路促进内皮细胞血管生成。

肿瘤和周围基质细胞之间的旁分泌信号对于卵巢癌进展过程中肿瘤微环境的维持至关重要。小细胞外囊泡（sEV；特别是外泌体）是由包括肿瘤细胞在内的许多细胞主动分泌的纳米级囊泡，被发现通过穿梭功能性 RNA 在细胞间通讯中发挥重要作用。尽管 microRNA（也称为 miRNA 或 miR）是一种调节基因表达的小非编码 RNA，可选择性地积聚在肿瘤 sEV 中并可介导细胞间通讯，但外泌体 miRNA 在卵巢肿瘤血管生成中发挥作用的确切生物学机制仍不清楚。在这项研究中，使用 ExoQuick 外泌体沉淀溶液从人卵巢癌细胞系 SKOV-3 的条件培养基中分离出 sEV，并通过扫描电子显微镜、动态光散射和免疫印迹进行表征。为了阐明对卵巢肿瘤细胞衍生的 sEVs (TD-sEVs) 可能产生的旁分泌作用，我们研究了内皮细胞中 TD-sEVs 触发的血管生成相关信号事件。由于 miR-141-3p 在卵巢肿瘤发病机制中的可能作用，我们将重点放在 miR-141-3p 上，与相应的供体细胞相比，检测到 miR-141-3p 在 TD-sEVs 中富集。我们发现 miR-141-3p 的 sEV 转移显着降低了细胞因子诱导型细胞因子信号转导抑制因子 (SOCS)-5 的表达水平，导致内皮细胞中 JAK-STAT3 通路上调。内皮细胞中的VEGFR-2导致促进内皮细胞迁移和血管生成。TD-sEVs 穿梭的 miR-141-3p 在调节 VEGFR-2 表达中的推定作用通过抗 miR-141-3p 挽救 TD-sEVs 对 VEGFR-2 表达的促进作用的能力得到证实。内皮细胞。我们的结果还表明，TD-sEVs 触发了内皮细胞中 NF-κB 信号的细胞内活性氧 (ROS) 依赖性激活。综上所述，我们的研究结果提出了一种新模型，其中上皮性卵巢癌分泌的 miR-141-3p 的 sEV 转移作为细胞间通讯的重要介质，促进内皮细胞血管生成。