低氧肺癌细胞衍生的外泌体 miR-21 通过靶向 IRF1 介导巨噬细胞 M2 极化并促进癌细胞增殖。
Hypoxic lung cancer cell-derived exosomal miR-21 mediates macrophage M2 polarization and promotes cancer cell proliferation through targeting IRF1.
机构信息
Department of Oncology, Xi'an traditional Chinese Medicine Hospital, Xi'an, 710021, Shanxi, China.
Department of Oncology, Xi'an Ninth Hospital, Xi'an, 710054, Shanxi, China.
出版信息
World J Surg Oncol. 2022 Jul 27;20(1):241. doi: 10.1186/s12957-022-02706-y.
BACKGROUND
Hypoxia is the hallmark of the tumor microenvironment (TME) and plays a critical role during the progress of tumor development. A variety of microRNAs (miRNAs) transmitted by tumor-derived exosomes were involved in intercellular communication. We aimed to elucidate the precise mechanism by which tumor cell-derived exosomes promote lung cancer development by affecting macrophage polarization under hypoxic conditions.
METHODS
CD163 signal in tumor tissue from lung cancer patients was detected by immunohistochemical (IHC). The M2 polarization-related markers were assessed by flow cytometry and western blot. Exosomes were isolated from normoxic and hypoxic lung cancer cell culture and characterized by transmission electron microscope (TEM), dynamic light scattering (DLS), and western blot. RNA sequencing was performed to show the abnormally expressed miRNAs in exosomes from normoxic and hypoxic lung cancer cell culture. In addition, CCK-8 and clone formation assays were used to assess cell proliferation. Dual luciferase reporter assay was used to evaluate the relationship between miR-21 and IRF1. For in vivo experiment, the male nude mice were injected with H1299 cells with exosomes and miR-21 mimic treatment.
RESULTS
Firstly, we found a strong CD163 signal in tumor tissue from lung cancer patients by IHC. Subsequently, we co-cultured lung cancer cell line H1299 with M0 macrophage THP-1 and found that H1299 in a hypoxic environment promoted THP-1 M2 polarization. PKH67 fluorescence staining experiments confirmed that exosomes of H1299 origin were able to enter THP-1 and induced M2 polarization. RNA sequencing of exosomes showed that miR-21 level was significantly higher in the hypoxic culture group compared to the normoxic group. Subsequent cellular assays showed that miR-21 inhibited the expression of IRF1 by targeting it. In addition, the overexpression of IRF1 reversed the role of miR-21 on macrophage M2 polarization. Finally, we have confirmed through animal experiments that either hypoxic environment or high miR-21 level promoted tumor progression.
CONCLUSIONS
High miR-21 level in hypoxic environments promoted macrophage M2 polarization and induced lung cancer progression through targeting IRF1.
背景
缺氧是肿瘤微环境（TME）的标志，在肿瘤发展过程中起着关键作用。肿瘤来源的外泌体传递的多种 microRNAs（miRNAs）参与细胞间通讯。我们旨在阐明肿瘤细胞衍生的外泌体在缺氧条件下通过影响巨噬细胞极化从而促进肺癌发展的确切机制。
方法
通过免疫组织化学（IHC）检测肺癌患者肿瘤组织中的 CD163 信号。通过流式细胞术和 Western blot 评估 M2 极化相关标志物。从常氧和缺氧肺癌细胞培养物中分离出外泌体，并通过透射电子显微镜（TEM）、动态光散射（DLS）和 Western blot 进行表征。进行 RNA 测序以显示常氧和缺氧肺癌细胞培养物中外泌体中异常表达的 miRNAs。此外，使用 CCK-8 和克隆形成测定法评估细胞增殖。双荧光素酶报告基因测定法用于评估 miR-21 和 IRF1 之间的关系。进行体内实验时，将带有外泌体和 miR-21 模拟物的 H1299 细胞注射到雄性裸鼠中。
结果
首先，我们通过 IHC 在肺癌患者的肿瘤组织中发现了强烈的 CD163 信号。随后，我们将肺癌细胞系 H1299 与 M0 巨噬细胞 THP-1 共培养，发现缺氧环境下的 H1299 促进了 THP-1 的 M2 极化。PKH67 荧光染色实验证实，源自 H1299 的外泌体能够进入 THP-1 并诱导 M2 极化。外泌体的 RNA 测序显示，与常氧组相比，缺氧培养组中外泌体的 miR-21 水平明显升高。随后的细胞实验表明，miR-21 通过靶向它抑制了 IRF1 的表达。此外，IRF1 的过表达逆转了 miR-21 对巨噬细胞 M2 极化的作用。最后，我们通过动物实验证实，无论是缺氧环境还是高 miR-21 水平都促进了肿瘤的进展。
结论
缺氧环境中高 miR-21 水平通过靶向 IRF1 促进巨噬细胞 M2 极化，并诱导肺癌进展。
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