Ku70去泛素化与乳腺癌转录激活因子2α稳定相关性研究  

Deubikuitylation of Ku70 leads to AP2α transcription factor high stability in breast cancer

作　　者：黄前川[1] 曹军皓[1] 丁进亚[1] 

机构地区：[1]广州军区武汉总医院检验科,湖北武汉430070

出　　处：《中华肿瘤防治杂志》2015年第3期174-178,共5页Chinese Journal of Cancer Prevention and Treatment

基　　金：湖北省青年基金(QJX2010-31)

摘　　要：目的探讨Ku70去泛素化与乳腺癌中转录激活因子2α(activatior protein 2α,AP-2α)蛋白稳定性增高的相关性。方法脉冲追踪技术分析AP-2α稳定性;免疫共沉淀验证AP-2α和Ku70在细胞内的相互作用;蛋白质印迹法检测蛋白酶体抑制剂MG132对乳腺癌和正常乳腺上皮细胞中AP-2α泛素化水平的影响;蛋白质印迹法检测乳腺癌组织标本中AP-2α及其泛素化水平;通过小干扰RNA(small interfering RNA,siRNA)抑制Ku70的表达,观察乳腺癌细胞中AP-2α泛素化、AP-2α和人表皮生长因子受体2(human epidermal growth factor receptor-2,HER-2)蛋白水平的变化。结果脉冲追踪结果显示,乳腺癌细胞系中AP-2α的蛋白半衰期32h,而正常乳腺上皮细胞仅2h,说明乳腺癌细胞系中AP-2α的蛋白稳定性增高;免疫共沉淀证实AP-2α和Ku70蛋白细胞内存在相互作用;蛋白质印迹检测显示,MG132提高正常乳腺上皮细胞AP-2α泛素化水平,但对乳腺癌细胞AP-2α的泛素化作用不明显;蛋白质印迹检测乳腺癌组织标本中AP-2α泛素化水平与其蛋白水平成负相关;siRNA抑制Ku70表达可以促进乳腺癌细胞中AP-2α泛素化,Spearman等级相关系数为-0.917,P<0.05;AP-2α及其靶基因HER-2的蛋白水平随处理时间增加而显著下降,Spearman等级相关系数为0.989,P=0.011。结论 Ku70可抑制乳腺癌转录因子AP-2α泛素化降解,稳定性增高的AP-2α在乳腺癌中呈高表达,从而激活癌基因HER-2。OBJECTIVE To investigate the effect of Ku70 deubikuitylation on the abnormally high stability of activatior protein 2α（AP-2α）transcription factor in breast cancer.METHODS The stability of AP-2α was investigated by pulse-chase method.The interaction between AP-2α and its interacting partner was tested by co-immunoprecipitation assay.The impact of proteasome inhibitor MG132 on the ubiquitylation level of AP-2α protein in breast cancer cell lines and mammary epithelial cell lines were determined by Western blot.The ubiquitination of AP-2α in breast tumor tissues was detected by Western blot.The ubiquitination of AP-2α and the level of AP-2α and human epidermal growth factor receptor-2（HER-2） in these cell lines were detected by Western blot following by Ku70 silenced with siRNA.RESULTS AP-2a protein with a half-life of over 32 hours in breast cancer cell lines was sharply higher than that of the 2 hours observed in mammary epithelial cell line,the stability of AP-2α protein in breast cancer cell lines was significantly improved.The interaction between AP-2α and Ku70 in cells was confirmed by co-immunoprecipitation assay.MG132 significantly increased the ubiquitylation level of AP-2α in mammary epithelial cell lines but not in breast cancer cell lines.The level of AP-2α was correlated negatively with ubiquitinated AP-2α in breast tumor tissues.In contrast to increase the level of of ubiquitinated AP-2α（Spearman correlation coefficient：-0.917,P＜ 0.05）,depletion of Ku70 leds to downregulation of AP-2α and HER2 expression in breast cancer cells（Spearman correlation coefficient：0.989,P＜0.05）.CONCLUSION In breast cancer cells and breast tumor tissues AP-2α is abnormally underubiquitinated caused by deubiquitin enzyme activity of Ku70,which blocks its degradation and increases its stability,as a result,HER-2 oncogene is constitutively transcripted and expressed.

关 键 词：乳腺肿瘤 KU70 泛素化 转录激活因子2α 人表皮生长因子受体2 

分 类 号：R737.9[医药卫生—肿瘤]