Department of Respiratory and Critical Care Medicine, Key Laboratory of Pulmonary Diseases of Health Ministry, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei 430030, China.
Department of Respiratory and Critical Care Medicine, Key Laboratory of Pulmonary Diseases of Health Ministry, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei 430030, China.
Life Sci. 2019 Mar 15;221:168-177. doi: 10.1016/j.lfs.2019.02.013. Epub 2019 Feb 7.
To investigate whether mucin 1 (MUC1) downregulation reduced the sensitivity of tumor necrosis factor-alpha (TNF-α)-induced bronchial epithelial cells to glucocorticoid-mediated necroptosis and explore the underlying mechanisms.
The human lung bronchial epithelial cell line (16HBE) was transfected with small interfering RNA (siRNA) against MUC1 and then stimulated by TNF-α, where some cells were pretreated with dexamethasone. Flow cytometry was performed to analyze necroptosis in 16HBE cells, and western blot analysis was used to detect protein expression levels of MUC1, glucocorticoid receptor (GR)α, GRβ, NF-κB p65, phospho-p65 (p-p65), and histone deacetylase-2 (HDAC2). Additionally, nuclear translocation of MUC1 and GRα was assessed by immunofluorescence.
We observed that MUC1 downregulation by siRNA significantly augmented TNF-α-induced necroptosis in 16HBE cells, and that dexamethasone showed impaired anti-necroptotic effects of MUC1 downregulation. Furthermore, we found that GRα nuclear translocation was inhibited in 16HBE cells with MUC1 downregulation, and that dexamethasone-mediated inhibition of p65 phosphorylation was lower in cells transfected with MUC1-siRNA compared to those transfected with negative control siRNA.
Impaired GRα nuclear translocation and inhibited p-p65 expression might contribute to glucocorticoid resistance caused by MUC1 deficiency in TNF-α-induced necroptosis in 16HBE cells, and should be considered as a potential target for the development of novel therapeutics for asthma.
研究黏蛋白 1（MUC1）下调是否降低了肿瘤坏死因子-α（TNF-α）诱导的支气管上皮细胞对糖皮质激素介导的坏死性凋亡的敏感性，并探讨其潜在机制。
用针对 MUC1 的小干扰 RNA（siRNA）转染人肺支气管上皮细胞系（16HBE），然后用 TNF-α刺激，其中一些细胞用地塞米松预处理。采用流式细胞术分析 16HBE 细胞的坏死性凋亡，并用 Western blot 分析检测 MUC1、糖皮质激素受体（GR）α、GRβ、核因子-κB p65、磷酸化 p65（p-p65）和组蛋白去乙酰化酶-2（HDAC2）的蛋白表达水平。此外，通过免疫荧光评估 MUC1 和 GRα 的核转位。
我们观察到 siRNA 下调 MUC1 显著增强了 16HBE 细胞中 TNF-α诱导的坏死性凋亡，地塞米松显示出对 MUC1 下调诱导的抗坏死性凋亡作用受损。此外，我们发现 16HBE 细胞中 MUC1 下调抑制了 GRα 的核转位，并且与转染阴性对照 siRNA 的细胞相比，转染 MUC1-siRNA 的细胞中 p65 磷酸化的地塞米松介导抑制作用较低。
GRα 核转位受损和 p-p65 表达抑制可能导致 TNF-α诱导的坏死性凋亡中 MUC1 缺乏引起的糖皮质激素抵抗，这可能成为开发新型哮喘治疗方法的潜在靶点。
