Department of Breast, Longhua Hospital Affiliated with Shanghai University of TCM, Shanghai 200032, China.
Department of Breast, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou 510006, China.
Curr Drug Metab. 2019;20(10):804-814. doi: 10.2174/1389200220666190819151946.
Chemoresistance blunts the therapeutic effect of cisplatin (DDP) on Triple-Negative Breast Cancer (TNBC). Researchers have not determined to date whether exosomes confer DDP resistance to other breast cancer cells or whether exosomal transfer of miRNAs derived from DDP-resistant TNBC cells confer DDP resistance.
The aim of this study was to investigate the role of exosomes in chemoresistance in breast cancer.
MDA-MB-231 cells resistant to DDP (231/DDP) were established. Exosomes were isolated from 231/DDP cells (DDP/EXO) and characterized by measuring the levels of protein markers, nanoparticle tracking analysis and transmission electron microscopy. MDA-MB-231, MCF-7 and SKBR-3 cell lines were treated with the isolated DDP/EXOs and cell proliferation and cytotoxicity to DDP were evaluated using MTT assays and apoptosis analyses. Western blotting was used to examine P-glycoprotein (P-gp) expression. Additionally, a microarray was used to analyse microRNA (miRNA) expression profiles in MDA-MB-231 and 231/DDP exosomes. The effects on miRNAs were determined using RT-PCR. Exosomal miR-423-5p was extracted, and differential expression was verified. The MTT cell viability assay, flow cytometry, and Transwell and immunofluorescence assays were performed to determine if differential expression of miR-423-5p sensitized cells to DDP in vitro.
Under a transmission electron microscope, the isolated exosomes exhibited a round or oval shape with a diameter ranging between 40 and 100 nm. DDP/EXOs labelled with PKH67 were taken up by MDA-MB-231 cells. After an incubation with DDP/EXOs, the cell lines exhibited a higher IC50 value for cisplatin, P-gp expression, migration and invasion capabilities and a lower apoptosis rate. Furthermore, 60 miRNAs from exosomes derived from 231/DDP cells were significantly up-regulated compared to exosomes from MDA-MB-231 cells. Notably, compared to the corresponding sensitive exosomes, miR-370-3p, miR-423-5p and miR-373 were the most differentially expressed miRNAs in DDP-resistant exosomes. We chose miR-423-5p, and up-regulation and down-regulation of exosomal miR-423-5p expression significantly affected DDP resistance.
Exosomes from DDP-resistant TNBC cells (231/DDP) altered the sensitivity of other breast cancer cells to DDP in an exosomal miR-423-5p dependent manner. Our research helps to elucidate the mechanism of DDP resistance in breast cancer.
化疗耐药削弱了顺铂（DDP）对三阴性乳腺癌（TNBC）的治疗效果。研究人员尚未确定外泌体是否赋予其他乳腺癌细胞对 DDP 的耐药性，或者来自 DDP 耐药性 TNBC 细胞的外泌体转移的 miRNA 是否赋予 DDP 耐药性。
本研究旨在探讨外泌体在乳腺癌化疗耐药中的作用。
建立 DDP 耐药的 MDA-MB-231 细胞（231/DDP）。从 231/DDP 细胞中分离出外泌体（DDP/EXO），并通过测量蛋白标志物、纳米颗粒跟踪分析和透射电子显微镜来进行表征。MDA-MB-231、MCF-7 和 SKBR-3 细胞系用分离的 DDP/EXO 处理，并用 MTT 分析和凋亡分析评估细胞增殖和对 DDP 的细胞毒性。Western blot 用于检测 P-糖蛋白（P-gp）表达。此外，使用微阵列分析 MDA-MB-231 和 231/DDP 外泌体中的 microRNA（miRNA）表达谱。使用 RT-PCR 确定 miRNA 的影响。提取外泌体 miR-423-5p，并验证差异表达。通过 MTT 细胞活力测定、流式细胞术、Transwell 和免疫荧光测定，确定 miR-423-5p 的差异表达是否在体外使细胞对 DDP 敏感。
在透射电子显微镜下，分离的外泌体呈圆形或椭圆形，直径在 40 至 100nm 之间。用 PKH67 标记的 DDP/EXO 被 MDA-MB-231 细胞摄取。用 DDP/EXO 孵育后，细胞系对顺铂的 IC50 值更高，P-gp 表达、迁移和侵袭能力更高，凋亡率更低。此外，与 MDA-MB-231 细胞来源的外泌体相比，来自 231/DDP 细胞的外泌体中 60 个 miRNA 明显上调。值得注意的是，与相应的敏感外泌体相比，miR-370-3p、miR-423-5p 和 miR-373 是 DDP 耐药外泌体中表达差异最大的 miRNA。我们选择了 miR-423-5p，上调和下调外泌体 miR-423-5p 的表达显著影响 DDP 耐药性。
来自 DDP 耐药性 TNBC 细胞（231/DDP）的外泌体以依赖外泌体 miR-423-5p 的方式改变了其他乳腺癌细胞对 DDP 的敏感性。我们的研究有助于阐明乳腺癌中 DDP 耐药的机制。
