BACKGROUND A critical feature for fibroblasts differentiation into myofibroblasts is the expression of alpha-smooth muscle actin (α-SMA) during the tissue injury and repair process. The epigenetic mechanism, DNA methylation, is involved in regulating α-SMA expression. It is not clear how methyl-CpG-binding protein 2 (MeCP2) interacts with CpG-rich region in α-SMA, and if the CpG methylation status would affect MeCP2 binding and regulation of α-SMA expression. METHODS The association of MeCP2 with α-SMA CpG rich region were examined by chromatin immunoprecipitation (ChIP) assays in primary fibroblasts from idiopathic pulmonary fibrosis (IPF) and non-IPF control individuals, and in the lung fibroblasts treated with profibrotic cytokine transforming growth factor β1 (TGF-β1). The regulation of α-SMA by MeCP2 was examined by knocking down MeCP2 with small interfering RNA (siRNA). To explore the effects of the DNA methylation status of the CpG rich region on α-SMA expression, the cells were treated with DNA methyltransferase inhibitor, 5'-azacytidine (5'-aza). The expression of α-SMA was examined by Western blot and quantitative polymerase chain reaction, the association with MeCP2 was assessed by ChIP assays, and the methylation status was checked by bisulfate sequencing. RESULTS The human lung fibroblasts with increased α-SMA showed an enriched association of MeCP2, while knockdown MeCP2 by siRNA reduced α-SMA upregulation by TGF-β1. The 5'-Aza-treated cells have decreased α-SMA expression with reduced MeCP2 association. However, bisulfite sequencing revealed that most CpG sites are unmethylated despite the different expression levels of α-SMA after being treated by TGF-β1 or 5'-aza. CONCLUSION Our data indicate that the methyl-binding protein MeCP2 is critical for α-SMA expression in human lung myofibroblast, and the DNA methylation status at the CpG rich region of α-SMA is not a determinative factor for its inducible expression.

中文翻译：

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MeCP2 表观遗传调节人肺成纤维细胞中的 α-平滑肌肌动蛋白。

背景成纤维细胞分化成肌成纤维细胞的一个关键特征是在组织损伤和修复过程中α-平滑肌肌动蛋白(α-SMA)的表达。表观遗传机制 DNA 甲基化参与调节 α-SMA 表达。目前尚不清楚甲基-CpG 结合蛋白 2 (MeCP2) 如何与 α-SMA 中富含 CpG 的区域相互作用，以及 CpG 甲基化状态是否会影响 MeCP2 结合和 α-SMA 表达的调节。方法 通过染色质免疫沉淀 (ChIP) 分析在来自特发性肺纤维化 (IPF) 和非 IPF 对照个体的原代成纤维细胞中以及在用促纤维化细胞因子转化生长因子处理的肺成纤维细胞中检测 MeCP2 与富含 α-SMA CpG 区域的关联β1 (TGF-β1)。通过用小干扰 RNA (siRNA) 敲低 MeCP2 来检查 MeCP2 对 α-SMA 的调节。为了探索富含 CpG 区域的 DNA 甲基化状态对 α-SMA 表达的影响，用 DNA 甲基转移酶抑制剂 5'-氮杂胞苷 (5'-aza) 处理细胞。通过蛋白质印迹和定量聚合酶链反应检查 α-SMA 的表达，通过 ChIP 测定评估与 MeCP2 的关联，并通过硫酸氢盐测序检查甲基化状态。结果 具有增加的 α-SMA 的人肺成纤维细胞显示出 MeCP2 的富集关联，而通过 siRNA 敲低 MeCP2 减少了 TGF-β1 对 α-SMA 的上调。5'-Aza 处理的细胞降低了 α-SMA 的表达，同时降低了 MeCP2 的结合。然而，亚硫酸氢盐测序显示，尽管经 TGF-β1 或 5'-aza 处理后 α-SMA 的表达水平不同，但大多数 CpG 位点未甲基化。结论 我们的数据表明甲基结合蛋白 MeCP2 对人肺肌成纤维细胞中 α-SMA 的表达至关重要，并且 α-SMA 富含 CpG 区域的 DNA 甲基化状态不是其诱导表达的决定因素。