首页|期刊导航|中华医学杂志（英文版）|Reversal of MDR1 gene-dependent multidrug resistance using short hairpin RNA expression vectors
中华医学杂志（英文版）2005，Vol.118Issue(11)：893-902,10.
Reversal of MDR1 gene-dependent multidrug resistance using short hairpin RNA expression vectors
Reversal of MDR1 gene-dependent multidrug resistance using short hairpin RNA expression vectors
摘要
Abstract
Background RNA interference using short hairpin RNA (shRNA) can mediate sequence-specific inhibition of gene expression in mammalian cells. A vector-based approach for synthesizing shRNA has been developed recently. Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers multidrug resistance (MDR) to cancer cells. In this study, we reversed MDR using shRNA expression vectors in a multidrug-resistant human breast cancer cell line (MCF-7/AdrR). Methods The two shRNA expression vectors were constructed and introduced into MCF-7/AdrR cells. Expression of MDR1 mRNA was assessed by RT-PCR, and P-gp expression was determined by Western Blot and immunocytochemistry. Apoptosis and sensitization of the breast cancer cells to doxorubicin were quantified by flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. Cellular daunorubicin accumulation was assayed by laser confocal scanning microscopy (LCSM). Statistical significance of differences in mean values was evaluated by Student's t tests. P<0.05 was considered statistically significant.Results In MCF-7/AdrA cells transfected with MDR1-A and MDR1-B shRNA expression vectors, RT-PCR showed that MDR1 mRNA expression was reduced by 40.9% (P<0.05), 30.1% (P<0.01) (transient transfection) and 37.6 % (P<0.05), 28.0% (P<0.01) (stable transfection), respectively. Western Blot and immunocytochemistry showed that P-gp expression was significantly and specifically inhibited. Resistance against doxorubicin was decreased from 162-fold to 109-fold (P<0.05), 54-fold (P<0.01) (transient transfection) and to 108-fold (P<0.05), 50-fold (P<0.01) (stable transfection). Furthermore, shRNA vectors significantly enhanced the cellular daunorubicin accumulation. The combination of shRNA vectors and doxorubicin significantly induced apoptosis in MCF-7/AdrR cells. Conclusions shRNA expression vectors effectively reduce MDR expression in a sustained fashion and can restore the sensitivity of drug-resistant cancer cells to conventional chemotherapeutic agents.关键词
multi-drug resistance/MDR/gene therapy/breast neoplasm/shRNAKey words
multi-drug resistance/MDR/gene therapy/breast neoplasm/shRNA分类
医药卫生引用本文复制引用
GAN Hui-zhu,ZHENG De-ming,ZHANG Gui-zhen,ZHAO Ji-sheng,ZHANG Feng-chun,BU Li-sha,YANG Shao-juan,PIAO Song-lan,DU Zhen-wu,GAO Shen..Reversal of MDR1 gene-dependent multidrug resistance using short hairpin RNA expression vectors[J].中华医学杂志（英文版）,2005,118(11):893-902,10.基金项目
This study was supported by research grants from National Natural Science Foundation of China (No. 30171064), and Research Development Foundation of Jilin University (No. 2003CX045). （No. 30171064）
