使用短发夹RNA表达载体逆转MDR1基因依赖性多药耐药性

Reversal of MDR1 gene-dependent multidrug resistance using short hairpin RNA expression vectors.

作者信息

Gan Hui-zhu, Zhang Gui-zhen, Zhao Ji-sheng, Zhang Feng-chun, Bu Li-sha, Yang Shao-juan, Piao Song-lan, Du Zhen-wu, Gao Shen, Zheng De-ming

机构信息

Central Research Department, China-Japan Union Hospital, Jilin University, Changchun 130031, China.

出版信息

Chin Med J (Engl). 2005 Jun 5;118(11):893-902.

DOI:

PMID:15978189

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.

摘要

背景

使用短发夹RNA（shRNA）的RNA干扰可介导哺乳动物细胞中基因表达的序列特异性抑制。最近已开发出一种基于载体的shRNA合成方法。P-糖蛋白（P-gp）是MDR1基因的产物，其过表达赋予癌细胞多药耐药性（MDR）。在本研究中，我们使用shRNA表达载体在多药耐药的人乳腺癌细胞系（MCF-7/AdrR）中逆转MDR。

方法

构建两种shRNA表达载体并将其导入MCF-7/AdrR细胞。通过RT-PCR评估MDR1 mRNA的表达，并通过蛋白质免疫印迹和免疫细胞化学测定P-gp的表达。分别通过流式细胞术和甲基噻唑基四氮唑（MTT）试验对乳腺癌细胞对阿霉素的凋亡和敏感性进行定量。通过激光共聚焦扫描显微镜（LCSM）测定细胞柔红霉素蓄积。通过学生t检验评估平均值差异的统计学意义。P <0.05被认为具有统计学意义。

结果

在转染了MDR1-A和MDR1-B shRNA表达载体的MCF-7/AdrA细胞中，RT-PCR显示MDR1 mRNA表达分别降低了40.9%（P <0.05）、30.1%（P <0.01）（瞬时转染）和37.6%（P <0.05）、28.0%（P <0.01）（稳定转染）。蛋白质免疫印迹和免疫细胞化学显示P-gp表达受到显著且特异性的抑制。对阿霉素的耐药性从162倍降至109倍（P <0.05）、54倍（P <0.01）（瞬时转染）以及降至108倍（P <0.05）、50倍（P <0.01）（稳定转染）。此外，shRNA载体显著增强了细胞柔红霉素的蓄积。shRNA载体与阿霉素的组合显著诱导了MCF-7/AdrR细胞的凋亡。