Background Hypoxia is the typical characteristic of keloids. The development of keloids is closely related to the abnormal phenotypic transition of macrophages. However, the role of exosomal microRNAs (miRNAs) derived from hypoxic macrophages in keloids remains unclear. This study aimed to explore the role of hypoxic macrophage-derived exosomes (HMDE) in the occurrence and development of keloids and identify the critical miRNA. Methods The expression of CD206+ M2 macrophage in keloids and normal skin tissues was examined through immunofluorescence. The polarization of macrophages under a hypoxia environment was detected through flow cytometry. The internalization of macrophage-derived exosomes in human keloid fibroblasts (HKFs) was detected using a confocal microscope. miRNA sequencing was used to explore the differentially expressed miRNAs in exosomes derived from the normoxic and hypoxic macrophage. Subsequently, the dual-luciferase reporter assay verified that phosphatase and tension homolog (PTEN) was miR-26b-5p's target. The biological function of macrophage-derived exosomes, miR-26b-5p and PTEN were detected using the CCK-8, wound-healing and Transwell assays. Western blot assay was used to confirm the miR-26b-5p's underlying mechanisms and PTEN-PI3K/AKT pathway. Results We demonstrated that M2-type macrophages were enriched in keloids and that hypoxia treatment could polarize macrophages toward M2-type. Compared with normoxic macrophages-derived exosomes (NMDE), HMDE promote the proliferation, migration and invasion of HKFs. A total of 38 differential miRNAs (18 upregulated and 20 downregulated) were found between the NMDE and HMDE. miR-26b-5p was enriched in HMDE, which could be transmitted to HKFs. According to the results of the functional assay, exosomal miR-26b-5p produced by macrophages facilitated HKFs' migration, invasion and proliferation via the PTEN-PI3K/AKT pathway. Conclusions The highly expressed miR-26b-5p in HMDE promotes the development of keloids via the PTEN-PI3K/AKT pathway.

中文翻译：

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缺氧巨噬细胞衍生的靶向 PTEN 的外泌体 miR-26b-5p 促进瘢痕疙瘩的发展。


背景 缺氧是疤痕疙瘩的典型特征。疤痕疙瘩的发生与巨噬细胞的异常表型转变密切相关。然而，源自缺氧巨噬细胞的外泌体 microRNA (miRNA) 在疤痕疙瘩中的作用仍不清楚。本研究旨在探讨缺氧巨噬细胞源性外泌体（HMDE）在瘢痕疙瘩发生发展中的作用并鉴定关键miRNA。方法采用免疫荧光法检测瘢痕疙瘩及正常皮肤组织中CD206+M2巨噬细胞的表达情况。通过流式细胞术检测缺氧环境下巨噬细胞的极化。使用共聚焦显微镜检测人瘢痕疙瘩成纤维细胞（HKF）中巨噬细胞衍生的外泌体的内化。 miRNA测序用于探索常氧和缺氧巨噬细胞来源的外泌体中差异表达的miRNA。随后，双荧光素酶报告基因测定证实磷酸酶和张力同源物（PTEN）是 miR-26b-5p 的靶标。使用 CCK-8、伤口愈合和 Transwell 检测检测巨噬细胞来源的外泌体、miR-26b-5p 和 PTEN 的生物学功能。 Western blot检测用于确认miR-26b-5p的潜在机制和PTEN-PI3K/AKT通路。结果我们证明M2型巨噬细胞在疤痕疙瘩中富集，缺氧治疗可以使巨噬细胞极化为M2型。与常氧巨噬细胞衍生的外泌体（NMDE）相比，HMDE 促进 HKF 的增殖、迁移和侵袭。在 NMDE 和 HMDE 之间总共发现了 38 个差异 miRNA（18 个上调，20 个下调）。 miR-26b-5p 在 HMDE 中富集，可传播至 HKF。 根据功能测定的结果，巨噬细胞产生的外泌体miR-26b-5p通过PTEN-PI3K/AKT途径促进HKF的迁移、侵袭和增殖。结论 HMDE中高表达的miR-26b-5p通过PTEN-PI3K/AKT通路促进瘢痕疙瘩的发生。