南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (8): 1166-1173.doi: 10.12122/j.issn.1673-4254.2022.08.08

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FUT8调控TGF-β1介导的肺成纤维细胞纤维化的机制

高崴崴，刘待见，张晓萍，冯青青，刘 颖   

  1. 郑州大学第二附属医院呼吸与危重症医学科，河南 郑州 450000
  • 出版日期:2022-08-20 发布日期:2022-09-05

FUT8 modulates galectin-3 expression to regulate TGF-β1-mediated fibrosis of lung fibroblasts

GAO Weiwei, LIU Daijian, ZHANG Xiaoping, FENG Qingqing, LIU Ying   

  1. Department of Respiratory and Critical Care Medicine, Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China
  • Online:2022-08-20 Published:2022-09-05

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摘要： 目的 探讨α-1，6岩藻糖基转移酶（FUT8）对人胚肺成纤维细胞（MRC-5）增殖、迁移和纤维化的作用及可能机制。方法 将24只C57/BL6小鼠随机分为对照组、博莱霉素组、sh-NC组和sh-FUT8组，应用Masson染色观察肺纤维化情况。细胞实验分别设置对照组：MRC-5正常培养；TGF-β1组：TGF-β1处理MRC-5；si-NC组：si-NC转染MRC-5后TGF-β1处理；si-FUT8组：si-FUT8转染MRC-5后TGF-β1处理；Gal-3组：si-FUT8和pcDNA3.1-Gal转染MRC-5后TGF-β1处理。CCK-8和BrdU方法检测细胞增殖；划痕实验检测细胞迁移；RT-qPCR和Western blot检测α-平滑肌肌动蛋白（α-SM A）、I型胶原蛋白（COLIA1）和细胞外基质纤维连接蛋白（FN）的表达水平。同时，免疫共沉淀检测了FUT8与半乳糖凝集素-3（Gal-3）的作用及沉默FUT8对FAK/Akt信号通路的调节。结果 沉默FUT8显著降低博莱霉素诱导的小鼠肺组织细胞外胶原沉积。沉默FUT8抑制TGF-β1介导的细胞增殖（186.81±6.29 vs 118.09±9.48，P<0.05）和迁移。FUT8缺失下调α-SMA、COLIA1和FN的mRNA和蛋白表达水平（P<0.05）。此外，FUT8可直接与Gal-3作用。沉默FUT8下调Gal-3的表达并抑制FAK/Akt信号通路，过表达Gal-3逆转FUT8对细胞增殖、迁移和纤维化的作用（P<0.05）。结论 FUT8通过调控Gal-3调控TGF-β1介导的MRC-5细胞增殖、迁移和纤维化，FAK/Akt信号通路可能参与了这个过程。

关键词: FUT8；galectin-3；增殖；迁移；纤维化

Abstract: Objective To investigate the regulatory role of α-1,6-fucosyltransferase (FUT8) in TGF-β1-induced proliferation, migration and fibrosis of human embryonic lung fibroblasts (MRC-5 cells) and explore the underlying molecular mechanism. Methods C57/BL6 mice were randomized into 4 groups for treatment with saline (control group), bleomycin, bleomycin+sh-NC or bleomycin + sh-FUT8, and pulmonary fibrosis was observed using Masson staining. MRC-5 cells were transfected with si-NC, FUT8 siRNA (si-FUT8), or both si-FUT8 and a galectin-3 (Gal-3) overexpression plasmid (pcDNA3.1-Gal) prior to TGF-β1 treatment, and the changes in cell proliferation and migration were assessed using CCK-8 assay, BrdU assay, and wound healing assay; the changes in the expression levels of α-SMA, collagen I (COLIA1) and extracellular matrix fibronectin (FN) were detected with real-time quantitative PCR (RT-qPCR) and Western blotting. The interaction of FUT8 and Gal-3 was tested using coimmunoprecipitation (Co-IP) assay, and the effect of FUT8 silencing on Gal-3 and FAK/Akt signaling pathways was analyzed. Results FUT8 knockdown significantly reduced bleomycin-induced extracellular collagen deposition in the lung tissues of the mice. Silencing FUT8 obviously inhibited cell proliferation (P<0.05) and migration mediated by TGF-β1. FUT8 knockdown down-regulated the mRNA and protein levels of α-SMA, COLIA1 and FN (P<0.05) in the cells. Coimmunoprecipitation analysis showed that FUT8 interacted with Gal-3. Silencing FUT8 significantly down-regulated Gal-3 expression and inhibited the activation of the FAK/Akt signaling pathway (P<0.05). Overexpression of Gal-3 obviously reversed the effects of FUT8 silencing on cell proliferation, migration and fibrosis (P<0.05). Conclusion FUT8 regulates TGF-β1-induced proliferation, migration and fibrosis of MRC-5 cells by modulating Gal-3 expression, in which the FAK/Akt pathway may play a role.

Key words: FUT8; galectin-3; proliferation; migration; fibrosis