摘要
目的 探讨黑素瘤细胞A375细胞外信号调节激酶（ERK）通路与核因子κB（NF-κB）信号转导通路的交互作用.方法 将培养的A375细胞分为对照组、选择性ERK信号通路抑制剂U0126（10 μmol/L、5 μmol/L）处理组和选择性NF-κB通路抑制剂BMS-345541（10 μmol/L、5μmol/L）处理组,分别提取蛋白质和mRNA,用Western印迹、RT-PCR观察U0126抑制ERK通路后,NF-κB P65、p-IκBα蛋白及NF-κB P65mRNA的表达,观察BMS-345541抑制NF-κB通路后ERK1/2、p-ERK1/2蛋白及ERK1 mRNA的表达.结果 应用10 μmol/L、5μmol/L U0126抑制ERK通路后,胞核NF-κB P65蛋白表达（分别为0.60±0.04、0.56±0.06）均较对照组（1.54±0.15）减少（P＜0.01）,其表达量与药物浓度无显著相关性（P＞ 0.01）;p-IκBα蛋白的表达（0.90±0.05、0.70±0.02）均较对照组（0.61±0.03）增加（P＜0.01）,两药物浓度组之间的表达量差异有统计学意义（P＜ 0.01）;NF-κB P65 mRNA的表达（0.79±0.05,0.75±0.04）均较对照组（0.86±0.05）减少（P＜0.01）.应用10 μmol/L BMS-345541抑制NF-κB通路后,ERK1/2、p-ERK1/2蛋白及ERK1 mRNA的表达（0.73±0.07、0.75±0.09、1.51±0.02）较对照组减少（P＜0.01）,BMS-345541浓度为5μmol/L时,ERK1/2 、p-ERK 1/2蛋白和ERK1 mRNA的表达（0.94±0.11、0.99±0.04、1.62±0.03）较对照组的差异无统计学意义（均P＞0.05）.结论 NF-κB通路和ERK通路在黑素瘤A375细胞中存在交互作用.
Objective To investigate the cross-talk between extracellular signal-regulated kinase （ERK） and nuclear factor κB （NF-κB） signal transduction pathways in A375 human melanoma cells.Methods Cultured A375 cells were randomly divided into 5 groups：control group receiving no treatment,two U0126 （a selective inhibitor of the ERK signaling pathway） groups treated with U0126 of 10 and 5 μmol/L,and two BMS-345541 groups treated with BMS-345541 of 10 and 5 μmol/L.After 24-hour treatment,Western blot and reverse transcription PCR were performed to measure the protein expressions of NF-κB P65,phosphorylated IκBα （p-IκBα）,ERK1/2,as well as p-ERK1/2,and the mRNA expressions of NF-κB P65 and ERK1,respectively.One-way analysis of variance and least significant difference （LSD）-t test were carried out for statistical analysis.Results After 24 hours of treatment with U0126 of 10 and 5 μmol/L,a significant decrease was noted in the relative expression level of NF-κB p65 protein （0.60 ± 0.04 and 0.56 ± 0.06 vs.1.54 ± 0.15,both P＜ 0.01） and mRNA （0.79 ± 0.05 and 0.75 ± 0.04 vs.0.86 ± 0.05,both P ＜ 0.01）,but a statistical increase in that of p-IκBα protein （0.90 ± 0.05 and 0.70 ± 0.02 vs.0.61 ± 0.03,both P ＜ 0.01） in the two U0126 groups compared with the control group; significant differences were observed in the expression level of p-IκBo protein （P ＜ 0.01） but not in that of NF-κB p65 protein （P ＞ 0.01） between the two U0126 groups.The relative expression levels of ERK1/2 and p-ERK1/2 proteins as well as ERK1 mRNA were significantly higher in the control A375 cells than those in the cells treated with BMS-345541 of 10 μmol/L （0.73 ± 0.07,0.75 ± 0.09,1.51 ± 0.02,all P ＜ 0.01）,but similar to those treated with BMS-345541 of 5 μmol/L （0.94 ± 0.11,0.99 ± 0.04,1.62 ± 0.03,all P ＞ 0.05）.Conclusion There is a cross-talk between ERK and NF-κB signal transduction pathways in A375 melanoma cells.
出处
《中华皮肤科杂志》
CAS
CSCD
北大核心
2014年第8期570-573,共4页
Chinese Journal of Dermatology
基金
山东省自然科学基金（ZR2010HM022）
山东省科技发展计划（2011YD18012）
