摘要
目的:研究Ku70与人肺腺癌耐药的相关性,观察调控Ku70表达能否逆转人肺腺癌耐药细胞系(A549/DDP)的耐药性。方法:采用RT-PCR、Western Blotting方法比较Ku70在人肺腺癌亲代细胞(A549)与耐药细胞系(A549/DDP)的表达水平。转染靶向Ku70基因siRNA(s-i Ku70)的A549/DDP细胞作为实验组,转染非特异性siRNA(s-i Scramble)的A549/DDP细胞作为阴性对照组,同时设空白组(A549/DDP细胞)。加药组在转染后48 h给予顺铂。应用MTT法检测各组细胞在顺铂作用下的生存率,并计算半数抑制浓度(IC50),采用流式细胞仪定量检测细胞凋亡率;采用分光光度法检测Caspase-3活化程度。结果:Ku70 mRNA和蛋白在A549/DDP细胞表达水平显著高于其亲代A549细胞(P<0.01);靶向Ku70的siRNA(s-i Ku70)明显下调A549/DDP细胞的Ku70mRNA和蛋白表达水平(P<0.01);在不同浓度顺铂作用24 h后,实验组细胞的生存率明显低于阴性对照组和空白组(P<0.01)。6 mg.L-1顺铂作用24 h后,实验组细胞凋亡率及Caspase-3活化程度高于阴性对照组和空白组(P<0.05)。结论:Ku70在人耐药肺腺癌细胞呈现高表达,提示Ku70参与肺腺癌耐药的形成;靶向Ku70siRNA导入人肺腺癌耐药细胞可特异性下调Ku70的表达,促进肺腺癌耐药细胞凋亡,逆转其耐药性,Ku70可能成为逆转肺癌耐药的一个新的分子靶点。
Objective To investigate the role of Ku70 in the chemoresistance of lung cancer and the potential of Ku70 small-interfering RNA（siRNA） as a therapy for reversal of cisplatin resistance in lung adenocarcinoma.Methods The Ku70 mRNA and protein expression levels were dectected by RT-PCR and Western blotting methods in human lung adenocarcinoma cells A549 and their cisplatin-resistant variant A549/DDP.The A549/DDP cells were divided into 3 groups：blank control,negative control group in which the cells were tranfected with nonspecific si-RNA（si-Scramble） and experiment group in which the cells were transfected with Ku70-specific siRNA（si-Ku70）.The cells in negative control group and experiment group were transfected 48 h prior to treatment with cisplatin.The cell survival was assessed by MTT assay.The apoptotic rate was determined by flow cytometry.The Caspase-3 activity was detected by spectrophotometer.Results The Ku70 mRNA and protein expressions in A549/DDP cells were significantly elevated compared with the parental A549 cells（P〈0.01）.The si-Ku70 down-regulated the mRNA and protein expressions of Ku70 in A549/DDP cells.After treated with different concentrations of cisplatin for 24 h,the cell viabilities of si-Ku70 cells were decreased significantly compared with control group（P〈0.01）.After treatment with 6 mg·L^-1 cisplatin for 24 h,the apoptotic rates and the activites of Caspase-3 in experiment group were increased significantly compared with control group（P〈0.01）.Conclusion The overexpression of Ku70 in cisplatin-resistant human lung adenocarcinoma cells suggests that Ku70 might be involved in the chemoresistance of lung cancer.Ku70-specific siRNA can down-regulate the expression of Ku70,promte apoptosis and reverse resistance to cisplatin in cisplatin-resistant A549/DDP cells.Ku70 may be a new target for reversal of chemoresistance of lung cancer.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2012年第2期240-244,共5页
Journal of Jilin University：Medicine Edition
基金
教育部博士学科点专项科研基金资助课题(200801830069)
关键词
肺腺癌
耐药性
顺铂
KU70
细胞凋亡
lung adenocarcinoma
drug resistance
cisplatin
Ku70
apoptosis
