靶向Ku70基因siRNA对人肺腺癌细胞顺铂耐药性的逆转作用

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（si-Ku70）的A549/DDP细胞作为实验组，转染非特异性siRNA（si-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（si-Ku70)明显下调A549/DDP细胞的Ku70 mRNA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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。

Reversal effect of Ku70-targeted siRNA on chemoresistance to cisplatin in human lung adenocarcinoma cells

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.