Objective: 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). Methods: 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. Results: 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 P<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 P<0.01]. Control siRNA transfection had no effect on HuR expression [mRNA level (1.02±0.10), protein level (0.97±0.06), all P>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 P>0.05). Conclusions: 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.
目的： 观察烟草烟雾提取物(CSE)刺激的小气道上皮中人抗原R的表达情况，探讨人抗原R对上皮间质转化(EMT)关键转录因子snail的调控作用。 方法： 体外培养人小气道上皮细胞(HSAEpiC)并给予CSE刺激，按CSE浓度和时间分为对照组、1%-24 h组、3%-24 h组、1%-48 h组及3%-48 h组。应用实时荧光定量PCR法、Western blot法检测CSE刺激下人抗原R mRNA和蛋白水平。采用小RNA(siRNA)干扰技术特异性降低人抗原R表达，根据转染情况分为对照组、转染对照组及转染组，观察转染后mRNA和蛋白表达的变化，检测CSE刺激及人抗原R干扰时转录因子snail、上皮细胞标志物E-钙黏蛋白及间叶细胞标志物波形蛋白表达水平变化。 结果： CSE刺激后，人抗原R mRNA及蛋白水平表达均升高，1%-24 h组mRNA吸光度值从(1.12±0.04)升高到(1.26±0.05)，蛋白表达量从(1.17±0.06)升高到(1.42±0.06)；3%-48 h组mRNA吸光度值从(1.41±0.06)升高到(1.49±0.06)；蛋白表达量从(1.35±0.08)升高到(1.42±0.06)，组内前后比较差异有统计学意义(均P<0.05)。人抗原R siRNA转染后，mRNA[(0.33±0.06)，(1.02±0.10)]及蛋白表达水平明显降低[(0.46±0.07)，(0.97±0.06)，均P<0.01]；对照siRNA转染后人抗原R表达变化无统计学意义[mRNA：(1.02±0.10)，蛋白：(0.97±0.06)，均P>0.05]。3%CSE刺激48 h后，人抗原R(1.47±0.11)、snail(1.46±0.05)和波形蛋白(1.56±0.05)表达升高，E-钙黏蛋白(0.49±0.05)表达降低；人抗原R siRNA转染后，人抗原R(0.84±0.06)、snail(1.22±0.06)、波形蛋白(1.11±0.09)表达降低，E-钙黏蛋白表达升高(0.73±0.06，均P<0.05)。 结论： CSE促进了小气道上皮细胞中人抗原R的表达；人抗原R参与了EMT关键转录因子snail的调控过程，并可能通过该作用调控EMT进程。.
Keywords: Cigarette smoke extract; Epithelial cell; Epithelial-mesenchymal transition; Human antigen R.