LncRNA FEZF1-AS1调控特发性肺间质纤维化的机制研究

Mechanism of LncRNA FEZF1-AS1 regulating idiopathic pulmonary fibrosis

摘要:

目的 探讨FEZ家族锌指1-反义RNA 1（LncRNA FEZF1-AS1）对肺间质纤维化细胞增殖、迁移、侵袭能力及上皮-间充质相互作用（EMT）的影响及其作用机制。方法 将转化生长因子-β₁（TGF-β₁）作用于A549细胞，诱导肺间质纤维化细胞模型，将其分为空白对照组（A549细胞组）和模型组。Western blotting检测细胞E-钙黏蛋白（E-cadherin）、N-钙黏蛋白（N-cadherin）及波形蛋白（Vimentin）表达，观察模型复制是否成功；实时荧光定量聚合酶链反应（qRT-PCR）检测细胞LncRNA FEZF1-AS1和miR-200c-3p基因表达。根据实验目的和转染质粒不同，将A549细胞分为空白对照组（Blank组）、TGF-β₁ + Si LncRNA FEZF1-AS1 NC组、TGF-β₁ + Si LncRNA FEZF1-AS1组。CCK-8法检测细胞增殖能力；细胞划痕实验检测细胞迁移能力；Transwell检测细胞侵袭能力。Western blotting检测细胞E-cadherin、N-cadherin及Vimentin蛋白表达；qRT-PCR检测细胞LncRNA FEZF1-AS1和miR-200c-3p基因表达。结果 模型组E-cadherin蛋白相对表达量较空白对照组降低（P <0.05），N-cadherin及Vimentin蛋白相对表达量较空白对照组升高（P <0.05）；模型组LncRNA FEZF1-AS1 基因相对表达量较空白对照组升高（P <0.05），miR-200c-3p 基因相对表达量较空白对照组降低（P <0.05）。与Blank组比较，TGF-β₁ + Si LncRNA FEZF1-AS1 NC组细胞增殖、迁移、侵袭能力升高（P <0.05）；与TGF-β₁ + Si LncRNA FEZF1-AS1 NC组比较，TGF-β₁ + Si LncRNA FEZF1-AS1组细胞增殖、迁移、侵袭能力降低（P <0.05）。与TGF-β₁ + Si LncRNA FEZF1-AS1 NC组比较，TGF-β₁ + Si LncRNA FEZF1-AS1组LncRNA FEZF1-AS1基因相对表达量及N-cadherin、Vimentin蛋白相对表达量降低（P <0.05），E-cadherin蛋白相对表达量升高（P <0.05）；与TGF-β₁ + Si LncRNA FEZF1-AS1 NC组比较，TGF-β₁ + Si LncRNA FEZF1-AS1组和Blank组miR-200c-3p 基因相对表达量升高（P <0.05）。结论 LncRNA FEZF1-AS1通过抑制miR-200c-3p促进肺间质纤维化细胞增殖、迁移、侵袭及EMT过程。

Abstract:

Objective To investigate the effects of the long non-coding RNA FEZF1-AS1 (LncRNA FEZF1-AS1) on the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of cells in idiopathic pulmonary fibrosis (IPF) and its underlying mechanisms.Methods A549 cells were treated with Transforming growth factor-β1 (TGF-β₁) to induce a pulmonary fibrosis cell model, dividing them into a blank control group (A549 cell group) and a model group. Western blotting was used to measure the expression of E-cadherin, N-cadherin, and Vimentin to verify the model's success. Real-time quantitative polymerase chain reaction (qRT-PCR) was employed to measure the expression of LncRNA FEZF1-AS1 and microRNA-200c-3p (miR-200c-3p). Based on experimental goals and different transfection plasmids, A549 cells were divided into three groups: Blank, TGF-β₁ + Si LncRNA FEZF1-AS1 NC, and TGF-β₁ + Si LncRNA FEZF1-AS1. Cell proliferation was evaluated using the CCK-8 assay, migration was assessed with wound healing assays, and invasion capabilities were measured using Transwell assays. Western blotting and qRT-PCR were repeated to assess protein and gene expression changes.Results In the model group, the relative expression of E-cadherin decreased, while N-cadherin and Vimentin increased compared to the blank control (P <0.05). LncRNA FEZF1-AS1 was upregulated, and miR-200c-3p was downregulated in the model group compared to the control (P <0.05). Compared to the Blank group, cell proliferation, migration, and invasion were increased in the TGF-β₁ + Si LncRNA FEZF1-AS1 NC group (P <0.05). In contrast, these parameters were reduced in the TGF-β₁ + Si LncRNA FEZF1-AS1 group compared to the NC group (P <0.05). Also, this group showed decreased expression of LncRNA FEZF1-AS1, N-cadherin, Vimentin, and increased E-cadherin compared to the NC group (P <0.05). The expression of miR-200c-3p was higher in the TGF-β₁ + Si LncRNA FEZF1-AS1 and Blank groups compared to the NC group (P < 0.05).Conclusions LncRNA FEZF1-AS1 promotes the proliferation, migration, invasion, and EMT processes in pulmonary fibrosis cells by inhibiting miR-200c-3p.