[核苷酸切除修复基因着色性干皮病A组反义RNA对人肺腺癌细胞系A549顺铂敏感性的增强作用]

[Enhancement effect of nucleotide excision repair gene xeroderma pigmentosun group a antisense RNA on sensitivity of human lung adenocarcinoma cell line A549 to cisplatin].

作者信息

Fan Wei, Zhang Hai-Long, Wu Xiao-Ming

机构信息

Institute of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, P. R. China.

出版信息

Ai Zheng. 2005 Apr;24(4):403-7.

PMID:15820060

Abstract

BACKGROUND & OBJECTIVE: Enhanced nucleotide excision repair (NER) capacity is an important mechanism of drug-resistance of tumor cells. Xeroderma pigmentosun group A (XPA) gene plays a key role in early stage of NER pathway. This study was to explore correlation between down-regulation of XPA gene induced by antisense RNA transfection and sensitivity of human lung adenocarcinoma cell line A549 to cisplatin.

METHODS

A549 cells were stably transfected with XPA antisense RNA. Positive cell clones were selected by limiting dilution. Northern blot and Western blot were applied to evaluate mRNA and protein levels of XPA in positive 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 cisplatin-damaged A549 cells.

RESULTS

Six positive cell clones, AS1-AS6, were obtained, mRNA and protein levels of XPA were significantly decreased in AS3-AS6 cells. In dose-dependent experiment, the 50% inhibitory concentrations (IC(50)) of cisplatin to parental A549 cells and AS1-AS6 cells were 8.1, 7.6, 4.7, 3.2, 1.9, 2.8, and 4.1 mug/ml, respectively. Sensitivities of AS3-AS6 cells were significantly higher than that of parental A549 cells (F = 9.75, 9.14, 7.39, 8.91u P = 0.005, 0.006, 0.012, 0.006). In addition, mRNA level of XPA was positively correlated with IC(50) of cisplatin (r = 0.927, P = 0.003). Time-effect experiment also showed increases of sensitivity to cisplatin in AS3-AS6 cells. HCR assay showed that 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 cellular NER capacities (r = 0.854, 0.696, 0.858u P = 0.014, 0.082, 0.013).

CONCLUSION

The targeted inhibition of XPA by antisense strategy can significantly decrease mRNA level of XPA, reduce cellular NER capacity, and sensitize lung cancer cells to cisplatin.

摘要

背景与目的

增强的核苷酸切除修复（NER）能力是肿瘤细胞耐药的重要机制。着色性干皮病A组（XPA）基因在NER途径的早期起关键作用。本研究旨在探讨反义RNA转染诱导的XPA基因下调与人肺腺癌细胞系A549对顺铂敏感性之间的相关性。

方法

用XPA反义RNA稳定转染A549细胞。通过有限稀释法筛选阳性细胞克隆。应用Northern印迹和Western印迹评估阳性细胞克隆中XPA的mRNA和蛋白水平。通过MTT法评估A549细胞对顺铂的敏感性。采用宿主细胞再活化（HCR）试验评估顺铂损伤的A549细胞的NER能力。

结果

获得6个阳性细胞克隆，即AS1 - AS6，AS3 - AS6细胞中XPA的mRNA和蛋白水平显著降低。在剂量依赖性实验中，顺铂对亲本A549细胞和AS1 - AS6细胞的50%抑制浓度（IC50）分别为8.1、7.6、4.7、3.2、1.9、2.8和4.1μg/ml。AS3 - AS6细胞的敏感性显著高于亲本A549细胞（F = 9.75、9.14、7.39、8.9l；P = 0.005、0.006、0.012、0.006）。此外，XPA的mRNA水平与顺铂的IC50呈正相关（r = 0.927，P = 0.003）。时间效应实验也显示AS3 - AS6细胞对顺铂的敏感性增加。HCR试验表明AS3 - AS6细胞的NER能力降低。用40、200和1000 ng/ml顺铂处理时，XPA的mRNA水平与细胞NER能力呈正相关（r = 0.854、0.696、0.858；P = 0.014、0.082、0.013）。