Gu X M, Wang X G, Sun J, Wang N, Jiang S J
Department of Respiratory Medicine, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, China.
Zhonghua Jie He He Hu Xi Za Zhi. 2017 Jul 12;40(7):515-519. doi: 10.3760/cma.j.issn.1001-0939.2017.07.008.
To investigate the abundance of human antigen R (HuR) in small airway epithelial cells stimulated by cigarette smoke extract (CSE) as well as the role of HuR in mediating snail which is recognized as a key transcription factor in regulating epithelial-mesenchymal transition (EMT). Human small airway epithelial cells (HSAEpiC) cultured in vitro were exposed to cigarette smoke extract (CSE) to model COPD status. Real-time PCR and western blotting analysis were used in detecting HuR protein and mRNA expression in cells with CSE which were divided into 5 groups: a control group, a 1%-24 h group, a 3%-24 h group, a 1%-48 h group and a 3%-48 h group. Small interfering RNA (siRNA) transfection was used to decrease HuR abundance. HuR expression at both mRNA and protein levels was detected using Real-time PCR and Western blotting analysis respectively, and the experiment was divided into 3 groups: a control group, a transfection control group and a transfection group. Snail, E-cadherin and vimentin levels were determined using Western blotting test in cells with both CSE exposure and HuR siRNA transfection which were divided into three groups: control group, CSE group and CSE + transfection group. After CSE stimulation, HuR expression was increased at both mRNA and protein levels [mRNA 1%-24 h group (1.12±0.04), 3%-24 h group (1.41±0.06), 1%-48 h group (1.26±0.05), 3%-48 h group (1.49±0.06), protein 3%-24 h group (1.35±0.08), 1%-48 h group (1.17±0.06), 3%-48h group (1.42±0.06) all <0.05]. Compared with the control siRNA, after HuR siRNA transfection, HuR mRNA and protein levels were significantly reduced [mRNA level (0.33±0.06) vs (1.02±0.10), protein level (0.46±0.07) vs (0.97±0.06), all <0.01]. Control siRNA transfection had no effect on HuR expression [mRNA level (1.02±0.10), protein level (0.97±0.06), all >0.05]. After 48 h stimulation with 3% CSE, the expression of HuR (1.47±0.11), snail (1.46±0.05) and vimentin (1.56±0.05) increased and the expression of E-cadherin (0.49±0.05) decreased. After transfection and CSE stimulation, the expression of HuR (0.84±0.06), snail (1.22±0.06) and vimentin (1.11±0.09) decreased and the expression of E-cadherin (0.73±0.06) increased. (All >0.05). CSE promoted the expression of HuR in human small airway epithelial cells. HuR participated in the regulation process of EMT key transcription factor snail and might regulate EMT process by this action.
为研究香烟烟雾提取物(CSE)刺激下人小气道上皮细胞中人类抗原R(HuR)的丰度,以及HuR在介导蜗牛蛋白(Snail)中的作用,蜗牛蛋白被认为是调节上皮-间质转化(EMT)的关键转录因子。体外培养的人小气道上皮细胞(HSAEpiC)暴露于香烟烟雾提取物(CSE)以模拟慢性阻塞性肺疾病(COPD)状态。采用实时荧光定量PCR和蛋白质印迹分析检测CSE处理细胞中HuR蛋白和mRNA表达,CSE处理细胞分为5组:对照组、1%-24 h组、3%-24 h组、1%-48 h组和3%-48 h组。使用小干扰RNA(siRNA)转染降低HuR丰度。分别采用实时荧光定量PCR和蛋白质印迹分析检测HuR在mRNA和蛋白质水平的表达,实验分为3组:对照组、转染对照组和转染组。采用蛋白质印迹法检测CSE暴露和HuR siRNA转染细胞中蜗牛蛋白、E-钙黏蛋白和波形蛋白水平,分为3组:对照组、CSE组和CSE +转染组。CSE刺激后,HuR在mRNA和蛋白质水平均升高[mRNA 1%-24 h组(1.12±0.04),3%-24 h组(1.41±0.06),1%-48 h组(1.26±0.05),3%-48 h组(1.49±0.06),蛋白质3%-24 h组(1.35±0.08),1%-48 h组(1.17±0.06),3%-48 h组(1.42±0.06),均<0.05]。与对照siRNA相比,HuR siRNA转染后,HuR mRNA和蛋白质水平显著降低[mRNA水平(0.33±0.06)对(1.02±0.10),蛋白质水平(0.46±0.07)对(0.97±0.06),均<0.01]。对照siRNA转染对HuR表达无影响[mRNA水平(1.02±0.10),蛋白质水平(0.97±0.06),均>0.05]。3% CSE刺激48 h后,HuR(1.47±0.11)、蜗牛蛋白(1.46±0.05)和波形蛋白(1.56±0.05)表达增加,E-钙黏蛋白(0.49±0.05)表达降低。转染并CSE刺激后HuR(0.84±0.06)、蜗牛蛋白(1.22±0.06)和波形蛋白(1.11±0.09)表达降低,E-钙黏蛋白(0.73±0.06)表达增加。(均>0.05)。CSE促进人小气道上皮细胞中HuR的表达。HuR参与EMT关键转录因子蜗牛蛋白的调控过程,并可能通过该作用调节EMT进程。