ERCC1反义RNA降低肺癌细胞对苯并(a)芘所致DNA损伤的修复能力

Antisense ERCC1 RNA decreases the repair capability of damaged DNA in lung cancer cells induced by benzopyrene

目的 探讨核苷酸切除修复基因ERCC1在肺癌细胞修复苯并 (a)芘所致DNA损伤中的作用。方法 构建表达ERCC1反义RNA的重组质粒 ,Lipofectin转染肺癌A5 4 9细胞 ,潮霉素筛选出稳定转染的细胞克隆 ;噻唑蓝法检测 2 4h细胞生存力 ;NorthernBlot分析细胞ERCC1基因mRNA表达水平 ;单细胞凝胶电泳技术检测DNA损伤程度 ,每组计算 5 0个细胞损伤情况。结果 筛选出表达ERCC1反义RNA的 7个阳性克隆 ,其生长特性与亲本细胞差别无显著性 ;内源性mRNA表达水平不同程度降低 ,为亲本细胞的 12 %～ 86 % ;DNA损伤修复速度减慢 ,10 μmol/L苯并 (a)芘作用 2 4h后再孵育 2 4h ,修复程度为亲本细胞的 2 9%～ 71% ;相关分析表明DNA损伤修复程度与ERCC1mRNA水平显著相关。结论 ERCC1反义RNA降低肺癌细胞对苯并 (a)芘所致DNA损伤的修复能力。

Objective To investigate the effect of ERCC1 gene on the repair capability of damaged DNA in lung cancer A549 cells induced by benzopyrene. Methods Recombinant plasmid expressing ERCC1 antisense RNA was constructed and transfected into A549 cells by Lipofectin reagent.The stable-transfected cell colonies were selected by hygromycin.Cell viability was determined by the MTT assay.The level of ERCC1 mRNA was measured by Northern Blot analysis.Single cell gel electrophoresis assay was applied to determine the cellular DNA damage and fifty cells for each group were counted. Results Seven positive colonies expressing ERCC1 antisense RNA were screened.There was no growth rate difference between the antisense-transfected cells and the parental cells.The endogenous mRNA level in transfected colonies decreased in varied degrees,i.e.12%～86% of that of the parental cells in Northern Blot assay.After 24 h treatment of 10 μmol/l benzopyrene,the repair capability for DNA damage in transfected colonies was reduced to 29%～71% of that of the parental cells.Also,a statistically significant correlation was observed between expression of ERCC1 mRNA and repair capability ( r =0.84). Conclusion Antisense ERCC1 RNA decreased the repair capability for damaged DNA in lung cancer cells induced by benzopyrene.