Department of Anesthesiology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Taiwan.
Department of Plastic Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Taiwan.
Int J Med Sci. 2021 Jan 1;18(4):1058-1066. doi: 10.7150/ijms.52768. eCollection 2021.
The heterogeneity of exosome populations presents a great challenge to their study. The current study was designed to investigate the potential heterogeneity miRNA contents in circulating exosomes purified via different exosomal markers. In this study, exosomes from the serum of C57BL/6 mice after cecum ligation and perforation (CLP) or sham operation were isolated by precipitation using ExoQuick-TC and affinity purified with anti-Rab5b, anti-CD9, anti-CD31, and anti-CD44 antibodies using the Exo-Flow Exosome Capture kit to collect exosome subpopulations. RNA extracted from the exosomes isolated by ExoQuick-TC were profiled by next-generation sequencing (NGS). Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was also employed to determine the expression profiles of four representative exosomal miRNAs (mmu-miR-486-5p, mmu-miR-10a-5p, mmu-miR-143-3p, and mmu-miR-25-3p) selected from the NGS analysis. The results revealed that the expression patterns of these miRNAs in exosomes isolated by ExoQuick-TC as determined by RT-qPCR and NGS were similar, showing upregulation of mmu-miR-10a-5p and mmu-miR-143-3p but downregulation of mmu-miR-25-3p and mmu-miR-486-5p following CLP when compared to the levels in exosomes from sham control mice. However, their expression levels in the antibody-captured exosome subpopulations varied. The miRNAs in the exosomes captured by anti-Rab5b or anti-CD9 antibodies were more similar to those isolated by ExoQuick-TC than to those captured by anti-CD44 antibodies. However, there were no significant differences in these four miRNAs in CD31-captured exosomes. This study demonstrated that purification with different exosomal markers allows the collection of different exosome subpopulations with various miRNA contents. The results of this study demonstrate the heterogeneity of circulating exosomes and suggest the importance of stratifying exosome subpopulations when using circulating exosomes as biomarkers or investigating exosome function. In addition, this study also emphasized the necessity of using a consistent exosome marker across different samples as detecting biomarkers.
外泌体群体的异质性对外泌体的研究提出了巨大的挑战。本研究旨在探讨通过不同外泌体标志物纯化的循环外泌体中潜在的miRNA 含量的异质性。在这项研究中，通过使用 ExoQuick-TC 沉淀分离来自盲肠结扎和穿孔（CLP）或假手术的 C57BL/6 小鼠血清中的外泌体，并使用 Exo-Flow Exosome Capture 试剂盒用抗 Rab5b、抗 CD9、抗 CD31 和抗 CD44 抗体亲和纯化，以收集外泌体亚群。从 ExoQuick-TC 分离的外泌体中提取 RNA ，并进行下一代测序（NGS）分析。还采用实时定量逆转录聚合酶链反应（RT-qPCR）来确定从 NGS 分析中选择的四个代表性外泌体 miRNA（mmu-miR-486-5p、mmu-miR-10a-5p、mmu-miR-143-3p 和 mmu-miR-25-3p）的表达谱。结果表明，通过 RT-qPCR 和 NGS 确定的 ExoQuick-TC 分离的外泌体中这些 miRNA 的表达模式相似，与 sham 对照小鼠相比，CLP 后 mmu-miR-10a-5p 和 mmu-miR-143-3p 上调，但 mmu-miR-25-3p 和 mmu-miR-486-5p 下调。然而，它们在抗体捕获的外泌体亚群中的表达水平不同。用抗 Rab5b 或抗 CD9 抗体捕获的外泌体中的 miRNA 与用 ExoQuick-TC 分离的 miRNA 更相似，而与用抗 CD44 抗体捕获的 miRNA 不同。然而，在 CD31 捕获的外泌体中，这四个 miRNA 没有显著差异。本研究表明，用不同的外泌体标志物纯化可以收集具有不同 miRNA 含量的不同外泌体亚群。本研究结果表明循环外泌体的异质性，并提示在将循环外泌体用作生物标志物或研究外泌体功能时，对外泌体亚群进行分层的重要性。此外，本研究还强调了在不同样本中使用一致的外泌体标志物作为检测生物标志物的必要性。
