Department of Gynaecology and Obstetrics, Fujian Medical University Provincial Clinical Medical College, Fujian Provincial Hospital, Fuzhou, Fujian 350001, China.
Department of Ultrasonography, Fujian Medical University Provincial Clinical Medical College, Fujian Provincial Hospital, Fuzhou, Fujian 350001, China.
Biomed Pharmacother. 2022 Mar;147:112680. doi: 10.1016/j.biopha.2022.112680. Epub 2022 Feb 3.
This study aimed to explore the effects of endometriosis (EMS)-derived exosomes and miR-301a-3p on the polarization of macrophages and investigate the involved molecular mechanism. The exosomes were isolated from ectopic endometrial tissues of EMS patients and normal human serum (NHS). Results of transmission electron microscope and Nanoparticle Tracking Analysis showed that both EMS-exosomes and NHS-exosomes are about 80 nm microvesicles. Exosomal markers CD63 and TSG101 were abundantly expressed in both EMS-exosomes and NHS-exosomes. No negative marker Calnexin was detected in NHS-exosomes. A small amount of Calnexin was detected in EMS-exosomes. THP-1 cells differentiatee to macrophages by incubating with phorbol-12-myristate-13-acetate. Effects of the exosomes on the phagocytosis and polarization of macrophages were evaluated by PKH26 fluorescent labeling and flow cytometry, respectively. Compared with the NHS-exosomes group, the phagocytic capacity of macrophages was reduced and the polarization of macrophages to M2 macrophages was promoted after EMS-exosomes treatment. Results of western blot showed that compared with the NHS-exosomes group, the EMS-exosomes treatment significantly up-regulated the expression of phosphatidylinositol 3-kinase (PI3K) and down-regulated the expression of phosphatase and tensin homologue deleted on chromosome 10 (PTEN). miR-301a-3p mimic, negative control (NC) mimic, miR-301a-3p inhibitor and NC inhibitor were transfected into cells. Transfection efficiency was confirmed by RT-qPCR. Effects of the miR-301a-3p expression on the macrophages polarization and the expression of Arg-1, PTEN and PI3K in the macrophages were evaluated by flow cytometry and western blot, respectively. miR-301a-3p overexpression significantly enhanced the ability of EMS-exosomes-inducing M2 transformation of macrophages, promoted the expression of Arg-1 and PI3K, and inhibited the PTEN expression. miR-301a-3p inhibitor significantly reduced the expression of Arg-1 and PI3K and promoted the PTEN expression. In conclusion, EMS derived exosomal miR-301a-3p mediated macrophage polarization via regulating PTEN-PI3K axis.
本研究旨在探讨子宫内膜异位症（EMS）来源的外泌体和 miR-301a-3p 对巨噬细胞极化的影响，并探讨其涉及的分子机制。从 EMS 患者异位子宫内膜组织和正常人血清（NHS）中分离出外泌体。透射电子显微镜和纳米颗粒跟踪分析结果显示，EMS 外泌体和 NHS 外泌体均为约 80nm 的微囊泡。外泌体标记物 CD63 和 TSG101 在 EMS 外泌体和 NHS 外泌体中均大量表达。NHS 外泌体中未检测到负标记物 Calnexin。在 EMS 外泌体中检测到少量 Calnexin。用佛波醇 12-肉豆蔻酸 13-乙酸酯孵育 THP-1 细胞分化为巨噬细胞。通过 PKH26 荧光标记和流式细胞术分别评估外泌体对巨噬细胞吞噬作用和极化的影响。与 NHS-外泌体组相比，EMS-外泌体处理后巨噬细胞的吞噬能力降低，向 M2 巨噬细胞极化增强。Western blot 结果显示，与 NHS 外泌体组相比，EMS 外泌体处理显著上调了磷酸肌醇 3-激酶（PI3K）的表达，下调了第 10 号染色体缺失的磷酸酶和张力蛋白同源物（PTEN）的表达。转染 miR-301a-3p 模拟物、阴性对照（NC）模拟物、miR-301a-3p 抑制剂和 NC 抑制剂。通过 RT-qPCR 验证转染效率。通过流式细胞术和 Western blot 分别评估 miR-301a-3p 表达对巨噬细胞极化以及巨噬细胞中 Arg-1、PTEN 和 PI3K 表达的影响。miR-301a-3p 过表达显著增强了 EMS 外泌体诱导的巨噬细胞 M2 转化能力，促进了 Arg-1 和 PI3K 的表达，抑制了 PTEN 的表达。miR-301a-3p 抑制剂显著降低了 Arg-1 和 PI3K 的表达，促进了 PTEN 的表达。总之，EMS 来源的外泌体 miR-301a-3p 通过调节 PTEN-PI3K 轴介导巨噬细胞极化。
