METHODS: Primary ASM cells derived from four donors were transfected with CEBPD or non-targeting (NT) siRNA and exposed to vehicle control, budesonide (100 nM, 18 h), TNFα (10 ng/ml, 18 h), or both budesonide and TNFα. Subsequently, RNA-Seq was used to measure gene expression levels, and pairwise differential expression results were obtained for exposures versus vehicle and knockdown versus control conditions. Weighted gene co-expression analysis was performed to identify groups of genes with similar expression patterns across the various experimental conditions (i.e., CEBPD knockdown status, exposures).
RESULTS: CEBPD knockdown altered expression of 3037 genes under at least one exposure (q-value < 0.05). Co-expression analysis identified sets of 197, 152 and 290 genes that were correlated with CEBPD knockdown status, TNFα exposure status, and both, respectively. JAK-STAT signaling pathway genes, including IL6R and SOCS3, were among those influenced by both TNFα and CEBPD knockdown. Immunoblot assays revealed that budesonide-induced IL-6R protein expression and augmented IL-6-induced STAT3 phosphorylation levels were attenuated by CEBPD knockdown in ASM.
CONCLUSIONS: CEBPD modulates glucocorticoid responses in ASM, in part via modulation of IL-6 receptor signaling.
方法:用CEBPD或非靶向(NT)siRNA转染来自四个供体的原代ASM细胞,并暴露于媒介物对照,布地奈德(100nM,18h),TNFα(10ng/ml,18h),或布地奈德和TNFα。随后,RNA-Seq用于测量基因表达水平,对于暴露与媒介物和敲低与对照条件,获得了成对差异表达结果。进行加权基因共表达分析,以鉴定在各种实验条件下具有相似表达模式的基因组(即,CEBPD击倒状态,暴露)。
结果:CEBPD敲低在至少一次暴露下改变了3037个基因的表达(q值<0.05)。共表达分析确定了与CEBPD敲低状态相关的197、152和290组基因,TNFα暴露状态,两者,分别。JAK-STAT信号通路基因,包括IL6R和SOCS3,均受TNFα和CEBPD敲低的影响。免疫印迹分析显示,ASM中CEBPD敲低减弱了布地奈德诱导的IL-6R蛋白表达和增强的IL-6诱导的STAT3磷酸化水平。
结论:CEBPD调节ASM的糖皮质激素反应,部分通过调节IL-6受体信号传导。