短发夹RNA干扰表达质粒逆转人乳腺癌细胞MCF-7／ADR多药耐药的研究  

Reversal of multi-drug resistance in human breast cancer MCF-7/ADR cells by short hairpin RNA expression plasmids

作　　者：魏军民[1] 侯明[1] 李丽珍[1] 孔峰[2] 卞继峰[2] 

机构地区：[1]山东大学齐鲁医院肿瘤中心,济南250012 [2]山东大学医学院生物化学教研室,济南250012

出　　处：《山东大学学报（医学版）》2008年第4期349-352,共4页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金资助课题(30672434)

摘　　要：目的构建靶向多药耐药基因(MDR-1)的短发夹RNA(short hairpin loop RNA shRNA)干扰表达质粒,并探讨其逆转人乳腺癌细胞MCF-7/ADR多药耐药的作用效果。方法根据MDR-1基因表达序列设计有效的RNA干扰片断,构建并获得MDR-1基因特异的shRNA质粒表达载体pRNAT-U6.1/Neo-mdr1,采用lipofectin2000分别转染人乳腺癌耐药细胞MCF-7/ADR,利用G418筛选稳定表达的细胞克隆。利用RT-PCR检测MDR-1-RNA的表达,WesternBlotting检测MDR-1蛋白质的表达,MTT法检测耐药逆转效果,罗丹明123外排实验检测p-gp的转运功能。结果成功构建表达siRNA的质粒载体pRNAT-U6.1/Neo-mdr1,转染人乳腺癌耐药细胞MCF-7/ADR后48 h及G418筛选后1、2月,mdr1-RNA及蛋白质表达均呈明显下降,p-gp的转运功能明显提高,对阿霉素的敏感性增强,与耐药细胞及转入空白载体的细胞比较,差异有统计学意义(P均<0.05)。筛选后1、2月与转入48 h比较,差异无统计学意义(P>0.05)。结论shRNA干扰表达质粒pRNAT-U6.1/Neo-mdr1能够稳定、持久的抑制MDR-1基因,逆转乳腺癌细胞MCF-7/ADR对阿霉素的耐药性。Objective To construct shRNA plasmids which target the MDR1 gene and to investigate their reversal effect on the human breast cancer cell line MCF-7/ADR. Methods Oligonucleotides of the MDR-1 gene were designed based on the Gene Bank and the MDR-1 shRNA expression plasmid pRNAT-U6.1/Neo-mdr1 was constructed. The human breast cancer cell line MCF-7/ADR was tranfected with the MDR-1 shRNA expression plasmids by lipofectamine 2000, and a positive clone was selected by G418. MDR-1mRNA was assayed by RT-PCR and protein expression was determined by Western blotting. The P-gp function was determined by rhodamine 123 retention and the efficiency of MCF-7/ADR to ADM was determined by the MTT method. Results MDR-1 shRNA expression plasmids were successfully constructed . The MDR-1mRNA and protein expressions were significantly decreased 48 hours after transfection, 1 month and 2 months after selection by G418, and sensitivity to P-gp-transpertable drags was restored. The transporting function of P-gp was increased and the efficiency of MCF-7/ADR to ADM significantly reversed. Conclusions The shRNA expression plasmid pRNAT-U6.1/Neo-mdr1 can permanently inhibit MDR-1 gene stability. The sensitivity of MCF-7/ADR to adriamycin is reversed.

关 键 词：RNA干扰 基因 MDR-1 抗药性 肿瘤 细胞株 MCF-7/ADR 

分 类 号：R737.9[医药卫生—肿瘤]