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.
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。
Keywords: FUT8; fibrosis; galectin-3; migration; proliferation.