BACKGROUND Fibroblasts regulate tissue homeostasis and the balance between tissue repair and fibrosis. CCAAT/enhancer-binding protein alpha (CEBPA) is a key transcription factor that regulates adipogenesis. CEBPA has been shown to be essential for lung maturation, and deficiency of CEBPA expression leads to abnormal lung architecture. However, its specific role in lung fibroblast regulation and fibrosis has not yet been elucidated. METHODS Lung fibroblast CEBPA expression, pro-fibrotic and lipofibroblast gene expression were assessed by qRT-PCR. CEBPA gain and loss of function experiments were carried out to evaluate the role of CEBPA in human lung fibroblast activation with and without TGF-β1 treatment. Adipogenesis assay was used to measure the adiopogenic potential of lung fibroblasts. Finally, CRISPR activation system was used to enhance endogenous CEBPA expression. RESULTS We found that CEBPA gene expression is significantly decreased in IPF-derived fibroblasts compared to normal lung fibroblasts. CEBPA knockdown in normal human lung fibroblasts enhanced fibroblast pro-fibrotic activation and ECM production. CEBPA over-expression by transient transfection in IPF-derived fibroblasts significantly reduced pro-fibrotic gene expression, ECM deposition and αSMA expression and promoted the formation of lipid droplets measured by Oil Red O staining and increased lipofibroblast gene expression. Inhibition of the histone methyl transferase G9a enhanced CEBPA expression, and the anti-fibrotic effects of G9a inhibition were partially mediated by CEBPA expression. Finally, targeted CRISPR-mediated activation of CEBPA resulted in fibroblasts switching from fibrogenic to lipofibroblast states. CONCLUSIONS CEBPA expression is reduced in human IPF fibroblasts and its deficiency reduces adipogenic potential and promotes fibrogenic activation. CEBPA expression can be rescued via an inhibitor of epigenetic repression or by targeted CRISPR activation, leading to reduced fibrogenic activation.

中文翻译：

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通过 CRISPR 介导的 CEBPA 表达对成纤维细胞状态的靶向调节。

背景技术成纤维细胞调节组织稳态以及组织修复和纤维化之间的平衡。CCAAT/增强子结合蛋白α (CEBPA) 是调节脂肪生成的关键转录因子。CEBPA 已被证明对肺成熟至关重要，CEBPA 表达不足会导致肺结构异常。然而，其在肺成纤维细胞调节和纤维化中的具体作用尚未阐明。方法通过qRT-PCR评估肺成纤维细胞CEBPA表达、促纤维化和脂肪成纤维细胞基因表达。进行 CEBPA 获得和丧失功能实验以评估 CEBPA 在有和没有 TGF-β1 治疗的情况下在人肺成纤维细胞活化中的作用。脂肪生成测定用于测量肺成纤维细胞的脂肪生成潜力。最后，CRISPR激活系统用于增强内源性CEBPA表达。结果 我们发现，与正常肺成纤维细胞相比，IPF 衍生的成纤维细胞中 CEBPA 基因表达显着降低。正常人肺成纤维细胞中的 CEBPA 敲低增强了成纤维细胞的促纤维化激活和 ECM 的产生。通过瞬时转染在 IPF 衍生的成纤维细胞中过表达 CEBPA 显着降低了促纤维化基因表达、ECM 沉积和 αSMA 表达，并促进了油红 O 染色测量的脂滴形成并增加了脂肪成纤维细胞基因的表达。组蛋白甲基转移酶 G9a 的抑制增强了 CEBPA 的表达，而 G9a 抑制的抗纤维化作用部分由 CEBPA 表达介导。最后，靶向 CRISPR 介导的 CEBPA 激活导致成纤维细胞从纤维化状态转变为脂肪成纤维细胞状态。结论 CEBPA 在人 IPF 成纤维细胞中的表达降低，其缺乏会降低脂肪生成潜力并促进纤维化激活。CEBPA 表达可以通过表观遗传抑制抑制剂或靶向 CRISPR 激活来拯救，从而减少纤维化激活。