[反义ERCC1 RNA降低苯并[a]芘诱导的肺癌细胞中受损DNA的修复能力]

[Antisense ERCC1 RNA decreases the repair capability of damaged DNA in lung cancer cells induced by benzo[a]pyrene].

作者信息

Wu Xiao-ming, Zhou Yi-kai, Xu Shun-qing, Hao Qiao-ling, Ren Shu

机构信息

Institute of Environmental Medicine, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030, China.

出版信息

Zhonghua Yu Fang Yi Xue Za Zhi. 2003 May;37(3):167-70.

PMID:12880561

Abstract

OBJECTIVE

To investigate the effect of ERCC1 gene on the repair capability of damaged DNA in lung cancer A549 cells induced by benzo[a]pyrene.

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% approximately 86% of that of the parental cells in Northern Blot assay. After 24 h treatment of 10 micro mol/l benzo[a]pyrene, the repair capability for DNA damage in transfected colonies was reduced to 29% approximately 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 benzo[a]pyrene.

摘要

目的

探讨ERCC1基因对苯并[a]芘诱导的肺癌A549细胞损伤DNA修复能力的影响。

方法

构建表达ERCC1反义RNA的重组质粒，并用脂质体转染试剂将其转染至A549细胞。通过潮霉素筛选稳定转染的细胞集落。采用MTT法测定细胞活力。用Northern Blot分析检测ERCC1 mRNA水平。应用单细胞凝胶电泳试验检测细胞DNA损伤情况，每组计数50个细胞。

结果

筛选出7个表达ERCC1反义RNA的阳性集落。反义转染细胞与亲本细胞的生长速率无差异。转染集落中的内源性mRNA水平有不同程度下降，即在Northern Blot分析中约为亲本细胞的12%至86%。用10 μmol/l苯并[a]芘处理24小时后，转染集落对DNA损伤的修复能力降至亲本细胞的29%至71%。此外，观察到ERCC1 mRNA表达与修复能力之间存在统计学显著相关性（r = 0.84）。