Medical Examination Center, Luoyang Central Hospital, Zhengzhou University, No.288, Middle Zhongzhou Rd, Xigong District, Luoyang, 471009, China.
Department of Oncology, Zhengzhou Central Hospital, Zhengzhou, Henan, China.
J Exp Clin Cancer Res. 2019 Feb 8;38(1):62. doi: 10.1186/s13046-019-1027-0.
To investigate the lung cancer-promoting mechanism of mesenchymal stem cell-secreted extracellular vesicles (MSC-EV).
EV were isolated from culture media of human bone marrow-derived MSCs that were pre-challenged with or without hypoxia (referred to as H-EV and N-EV, respectively). After treatment with N-EV or H-EV, A549 and H23 cell proliferation, apoptosis, trans-well invasion and epithelial-to-mesenchymal transition (EMT) were examined. Polarization of human primary monocytes-derived macrophages with or without N-EV or H-EV induction were analyzed by flow cytometry and ELISA. PTEN, PDCD4 or RECK gene was overexpressed in A549 cells, while miR-21-5p was knocked down in MSCs, A549 or H23 lung cancer cells or primary monocytes by miR-21-5p inhibitor transfection. Protein level of PTEN, PDCD4, RECK, AKT or STAT3 as well as phosphorylation level of AKT or STAT3 protein were assayed by western blot. Tumorigenicity of A549 and H23 cells with or without MSC-EV co-injection was assayed on immunocompromised mice. The xenograft tumor were examined for cell proliferation, angiogenesis, apoptosis and intra-tumoral M1/M2 macrophage polarization.
Comparing to N-EV, H-EV treatment significantly increased A549 and H23 cell proliferation, survival, invasiveness and EMT as well as macrophage M2 polarization. MiR-21-5p knocked down significantly abrogated the cancer-promoting and macrophage M2 polarizing effects of H-EV treatment. H-EV treatment downregulated PTEN, PDCD4 and RECK gene expression largely through miR-21-5p. Overexpressing PTEN, PDCD4 and RECK in A549 cells significantly reduced the miR-21-5p-mediated anti-apoptotic and pro-metastatic effect of H-EV, while overexpressing PTEN in monocytes significantly reduced macrophage M2 polarization after induction with the presence of H-EV. H-EV co-injection significantly increased tumor growth, cancer cell proliferation, intra-tumoral angiogenesis and M2 polarization of macrophages in vivo partially through miR-21-5p.
Increased miR-21-5p delivery by MSC-EV after hypoxia pre-challenge can promote lung cancer development by reducing apoptosis and promoting macrophage M2 polarization.
研究间充质干细胞分泌的细胞外囊泡（MSC-EV）促进肺癌的机制。
从预先经历缺氧或未经历缺氧预处理的人骨髓来源间充质干细胞的培养上清液中分离 EV（分别称为 H-EV 和 N-EV）。用 N-EV 或 H-EV 处理 A549 和 H23 细胞后，检测细胞增殖、凋亡、Transwell 侵袭和上皮间质转化（EMT）。通过流式细胞术和 ELISA 分析人原代单核细胞来源的巨噬细胞经 N-EV 或 H-EV 诱导后的极化情况。通过 miR-21-5p 抑制剂转染，在 A549 细胞中过表达 PTEN、PDCD4 或 RECK 基因，在 MSC、A549 或 H23 肺癌细胞或原代单核细胞中敲低 miR-21-5p。通过 Western blot 检测 PTEN、PDCD4、RECK、AKT 或 STAT3 蛋白的表达水平以及 AKT 或 STAT3 蛋白的磷酸化水平。在免疫功能低下的小鼠中检测 A549 和 H23 细胞与 MSC-EV 共注射后的肿瘤生成能力。检测异种移植瘤中的细胞增殖、血管生成、凋亡和肿瘤内 M1/M2 巨噬细胞极化。
与 N-EV 相比，H-EV 处理显著增加了 A549 和 H23 细胞的增殖、存活、侵袭和 EMT 以及巨噬细胞 M2 极化。miR-21-5p 敲低显著消除了 H-EV 处理的促癌和促进巨噬细胞 M2 极化作用。H-EV 处理通过 miR-21-5p 使 PTEN、PDCD4 和 RECK 基因表达显著下调。在 A549 细胞中过表达 PTEN、PDCD4 和 RECK 显著降低了 H-EV 介导的 miR-21-5p 抗凋亡和促转移作用，而在单核细胞中过表达 PTEN 可在存在 H-EV 诱导的情况下显著减少巨噬细胞 M2 极化。H-EV 共注射显著增加了体内肿瘤生长、癌细胞增殖、肿瘤内血管生成和巨噬细胞 M2 极化。
缺氧预处理后的 MSC-EV 中 miR-21-5p 含量的增加可通过减少细胞凋亡和促进巨噬细胞 M2 极化来促进肺癌的发展。
