支气管肺泡灌洗液体外泌体中miR-204-5p通过AP1S2对肺纤维化进展的作用机制
Mechanism of miR-204-5p in exosomes derived from bronchoalveolar lavage fluid on the progression of pulmonary fibrosis via AP1S2.
作者信息
Zhu Liang, Chen Yahui, Chen Mo, Wang Wenwen
机构信息
Department of Rheumatism Immunology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Department of Rheumatism Immunology, Ningbo Sixth Hospital, Ningbo, China.
出版信息
Ann Transl Med. 2021 Jul;9(13):1068. doi: 10.21037/atm-20-8033.
BACKGROUND
Exosomes are nanoscale vesicles secreted by various types of cells that are responsible for intracellular communication. Despite that bronchoalveolar lavage fluid (BALF) has been proven to involve in tumor development, more efforts are required to investigate the impact of BALF on pulmonary fibrosis (PF). This study aimed to investigate the mechanism of how exosomal miR-204-5p from BALF facilitates PF progression in rats.
METHODS
PF rat model was established by intratracheal injection of bleomycin. BALF-derived exosomes (Exo) were extracted from normal and PF rats. PF-Exo (BALF-derived Exo from PF rats) and miR-204-5p antagomir were injected into rats to investigate the effect of exosomal miR-204-5p on PF. Collagen content in lung tissues of rats was assessed by Masson staining, hydroxyproline (HYP) content assay and immunohistochemistry (IHC). Primary lung fibroblasts were isolated, and treated by TGF-β1. After co-transfection of PF-Exo, miR-204-5p inhibitor and sh-AP1S2, cell proliferation, levels of miR-204-5p, fibrotic markers α-SMA and collagen 1 (Col 1), and proteins of autophagy markers LC3II, LC3I and P62 were measured. The interaction between miR-204-5p and AP1S2 was determined by bioinformatics online software TargetScan and dual-luciferase reporter assay.
RESULTS
miR-204-5p was abundantly expressed in the PF-Exo group. PF-Exo injection potentiated PF progression and proliferation ability of lung fibroblasts and . Injection with PF-Exo and miR-204-5p antagomir significantly increased the LC3II/I ratio and decreased the HYP content, proteins of α-SMA, Col 1 and P62, collagen content in rat lung tissues of PF rats. TGF-β1 induction elevated the LC3II/LC3I ratio, suppressed the cell proliferation rate, and decreased the levels of α-SMA, Col 1 and P62. Additionally, AP1S2 was a direct target of miR-204-5p. miR-204-5p inhibitor can counteract the effect of PF-Exo in proliferation of lung fibroblasts, while sh-AP1S2 eliminated the effect of miR-204-5p inhibitor.
CONCLUSIONS
Exosomal miR-204-5p from BALF inhibits autophagy to promote the progression of PF rats by targeting AP1S2.
背景
外泌体是由多种类型细胞分泌的纳米级囊泡，负责细胞内通讯。尽管支气管肺泡灌洗液（BALF）已被证明参与肿瘤发展，但仍需要更多努力来研究BALF对肺纤维化（PF）的影响。本研究旨在探讨BALF来源的外泌体miR-204-5p促进大鼠PF进展的机制。
方法
通过气管内注射博来霉素建立PF大鼠模型。从正常和PF大鼠中提取BALF来源的外泌体（Exo）。将PF-Exo（PF大鼠来源的BALF外泌体）和miR-204-5p拮抗剂注射到大鼠体内，以研究外泌体miR-204-5p对PF的影响。通过Masson染色、羟脯氨酸（HYP）含量测定和免疫组织化学（IHC）评估大鼠肺组织中的胶原蛋白含量。分离原代肺成纤维细胞，并用TGF-β1处理。在共转染PF-Exo、miR-204-5p抑制剂和sh-AP1S2后，测量细胞增殖、miR-204-5p水平、纤维化标志物α-SMA和胶原蛋白1（Col 1）以及自噬标志物LC3II、LC3I和P62的蛋白质水平。通过生物信息学在线软件TargetScan和双荧光素酶报告基因测定确定miR-204-5p与AP1S2之间的相互作用。
结果
miR-204-5p在PF-Exo组中大量表达。注射PF-Exo可增强PF进展和肺成纤维细胞的增殖能力。注射PF-Exo和miR-204-5p拮抗剂可显著提高PF大鼠肺组织中LC3II/I比值，降低HYP含量、α-SMA、Col 1和P62的蛋白质水平以及胶原蛋白含量。TGF-β1诱导可提高LC3II/LC3I比值，抑制细胞增殖率，并降低α-SMA、Col 1和P62的水平。此外，AP1S2是miR-204-5p的直接靶点。miR-204-5p抑制剂可抵消PF-Exo对肺成纤维细胞增殖的影响，而sh-AP1S2可消除miR-204-5p抑制剂的作用。
结论
BALF来源的外泌体miR-204-5p通过靶向AP1S2抑制自噬，促进PF大鼠的进展。
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