Department of Gastrointestinal Surgery, Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China.
Department of Hepatobiliary and Pancreatic Surgery, Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China.
Mol Cell Biol. 2021 Apr 22;41(5). doi: 10.1128/MCB.00517-20.
Exosomes are microvesicles secreted by body cells for intercellular communication. The circular RNA circ_0000338 was found to be present in extracellular vesicles and improve the chemoresistance of colorectal cancer (CRC) cells. However, the role of exosomal circ_0000338 in 5-fluorouracil (5-FU) resistance in CRC is largely unknown. The levels of circ_0000338, microRNA 217 (miR-217), and miR-485-3p were detected using quantitative real-time PCR (qRT-PCR). The 50% inhibitory concentration (IC) values of cells for 5-FU, cell proliferation, and apoptosis were evaluated using cell counting kit 8 (CCK-8), colony formation, flow cytometry, and Western blot assays. The interaction between miR-217 or miR-485-3p and circ_0000338 was confirmed by RNA immunoprecipitation (RIP), dual-luciferase reporter, and pulldown assays. Exosomes were isolated by ultracentrifugation and qualified by transmission electron microscopy (TEM), Nanosight tracking analysis (NTA), and Western blotting. Xenograft models were performed to analyze whether circ_0000338-loaded exosomes could increase resistance of CRC cells to 5-FU The circ_0000338 level was elevated in 5-FU-resistant CRC tissues and cells, and circ_0000338 knockdown sensitized 5-FU-resistant CRC cells to 5-FU through enhancing apoptosis and decreasing proliferation Mechanistically, circ_0000338 directly bound to miR-217 and miR-485-3p, and the inhibition of miR-217 or miR-485-3p reversed the effects of circ_0000338 knockdown on cell 5-FU resistance in CRC. Additionally, extracellular circ_0000338 could be incorporated into secreted exosomes and transmitted to 5-FU-sensitive cells. Treatment-sensitive cells with exosomes containing circ_0000338 reduced the 5-FU response in CRC both and Besides that, the exosomal circ_0000338 concentration was higher in patients exhibiting resistance to 5-FU and showed good diagnostic efficiency in 5-FU-resistant CRC. The delivery of circ_0000338 via exosomes enhanced 5-FU resistance in CRC through negative regulation of miR-217 and miR-485-3p, indicating a promising diagnostic and therapeutic marker for 5-FU-based chemotherapy in CRC patients.
外泌体是体细胞分泌的用于细胞间通讯的微小囊泡。circ_0000338 环状 RNA 被发现存在于细胞外囊泡中，并提高结直肠癌 (CRC) 细胞的化疗耐药性。然而，外泌体 circ_0000338 在 CRC 中对 5-氟尿嘧啶 (5-FU) 耐药性的作用在很大程度上尚不清楚。使用实时定量 PCR (qRT-PCR) 检测 circ_0000338、microRNA 217 (miR-217) 和 miR-485-3p 的水平。使用细胞计数试剂盒 8 (CCK-8)、集落形成、流式细胞术和 Western blot 测定评估细胞对 5-FU、细胞增殖和细胞凋亡的 50%抑制浓度 (IC) 值。通过 RNA 免疫沉淀 (RIP)、双荧光素酶报告基因和下拉测定证实了 miR-217 或 miR-485-3p 与 circ_0000338 之间的相互作用。通过超速离心分离外泌体，并通过透射电子显微镜 (TEM)、纳米粒子跟踪分析 (NTA) 和 Western blot 进行鉴定。进行异种移植模型分析以研究载有 circ_0000338 的外泌体是否可以增加 CRC 细胞对 5-FU 的耐药性。在 5-FU 耐药性 CRC 组织和细胞中 circ_0000338 水平升高，circ_0000338 敲低通过增强凋亡和减少增殖来使 5-FU 耐药性 CRC 细胞对 5-FU 敏感。在机制上，circ_0000338 直接与 miR-217 和 miR-485-3p 结合，抑制 miR-217 或 miR-485-3p 逆转了 circ_0000338 敲低对细胞的作用。此外，细胞外 circ_0000338 可以整合到分泌的外泌体中并传递给 5-FU 敏感细胞。含有 circ_0000338 的外泌体处理敏感细胞可降低 CRC 中 5-FU 的反应，无论是在体内还是体外。此外，对 5-FU 耐药的患者中外泌体 circ_0000338 的浓度更高，并在 5-FU 耐药性 CRC 中具有良好的诊断效率。通过外泌体递送 circ_0000338 通过负调控 miR-217 和 miR-485-3p 增强 CRC 中 5-FU 的耐药性，表明其在基于 5-FU 的化疗的 CRC 患者中具有有前途的诊断和治疗标志物。
