核苷酸切除修复基因XPA反义RNA增强肺癌细胞对顺铂的敏感性

Enhancement Effect of Nucleotide Excision Repair Gene Xeroderma Pigmentosun Group A Antisense RNA on Sensitivity of Human Lung Adenocarcinoma Cell Line A549 to Cisplatin

背景与目的:目前认为,肿瘤细胞自身核苷酸切除修复(nucleotideexcisionrepair,NER)能力增强是肿瘤细胞产生耐药性最重要的机制之一。着色性干皮病A(xerodermapigmentosungroupA,XPA)基因在核苷酸切除修复早期发挥核心作用。本研究拟探讨XPA基因表达与肺癌细胞株对顺铂敏感性的关系。方法:将XPA的反义RNA稳定转染人肺癌细胞株A549,用有限稀释法筛选阳性细胞克隆。分别用Northernblot和Westernblot法检测阳性细胞克隆XPAmRNA和蛋白水平;MTT法检测肿瘤细胞对顺铂敏感性;宿主细胞再活化反应(hostcellreactivation,HCR)检测肿瘤细胞DNA损伤修复能力。结果:筛选得到6个阳性克隆AS1～AS6,其中AS3～AS6细胞克隆的XPAmRNA和蛋白水平均明显降低。剂量依赖实验表明,顺铂对A549细胞与AS1～AS6细胞克隆的半数抑制浓度(IC50)分别为8.1、7.6、4.7、3.2、1.9、2.8、4.1滋g/ml。统计学分析表明,与A549细胞相比,AS3～AS6细胞对顺铂的敏感性明显增强(F=9.75、9.14、7.39、8.91,P=0.005、0.006、0.012、0.006),而且XPAmRNA表达水平与细胞IC50值呈显著相关(r=0.927,P=0.003)。处理24、48、72h后,AS3～AS6细胞对顺铂的敏感性同样显著增强。HCR实验结果表明,AS3～AS6细胞的NER能力显著减弱,而且XPAmRNA表达水平与细胞NER能力呈?

BACKGROUND & OBJECTIVE: Enhanced nucleotide excision repair (NER) ca pacity is an important mechanism of drug-resistance of tumor cells. Xeroderma pi gmentosun group A (XPA) gene plays a key role in early stage of NER pathway. Thi s study was to explore correlation between down-regulation of XPA gene induced b y antisense RNA transfection and sensitivity of human lung adenocarcinoma cell l ine A549 to cisplatin. METHODS: A549 cells were stably transfected with XPA anti sense RNA. Positive cell clones were selected by limiting dilution. Northern blo t and Western blot were applied to evaluate mRNA and protein levels of XPA in po sitive cell clones. Sensitivity of A549 cells to cisplatin was evaluated by MTT assay. Host cell reactivation (HCR) assay was used to assess NER capacity of cis platin-damaged A549 cells. RESULTS: Six positive cell clones, AS1-AS6, were obta ined, mRNA and protein levels of XPA were significantly decreased in AS3-AS6 cel ls. In dose-dependent experiment, the 50% inhibitory concentrations (IC50) of ci splatin to parental A549 cells and AS1-AS6 cells were 8.1, 7.6, 4.7, 3.2, 1.9, 2 .8, and 4.1 μg/ml, respectively. Sensitivities of AS3-AS6 cells were significan tly higher than that of parental A549 cells (F = 9.75, 9.14, 7.39, 8.91; P = 0.0 05, 0.006, 0.012, 0.006). In addition, mRNA level of XPA was positively correlat ed with IC50 of cisplatin (r = 0.927, P = 0.003). Time-effect experiment also sh owed increases of sensitivity to cisplatin in AS3-AS6 cells. HCR assay showed th at NER capacities of AS3-AS6 cells were reduced. When treated with 40, 200, and 1 000 ng/ml of cisplatin, mRNA levels of XPA were positively correlated with cel lular NER capacities (r = 0.854, 0.696, 0.858; P = 0.014, 0.082, 0.013). CONCLUS ION: The targeted inhibition of XPA by antisense strategy can significantly decr ease mRNA level of XPA, reduce cellular NER capacity, and sensitize lung cancer cells to cisplatin.