shRNA逆转耐阿霉素人乳腺癌细胞株（MCF-7/AdrR）的多药耐药性

目的〖HT5"SS〗： 利用短发夹RNA（short hairpin RNA,shRNA）表达载体逆转耐阿霉素人乳腺癌细胞株(MCF7/AdrR)的多药耐药性（multidrug resistance,MDR）。〖HT5W〗方法〖HT5"SS〗： 构建2个MDR1基因shRNA表达质粒,稳定转染MCF7/AdrR细胞。RTPCR分析MDR1基因mRNA的表达,Western blotting检测P糖蛋白(Pgp)的表达,流式细胞术和MTT法分别检测乳腺癌细胞的凋亡和对阿霉素的敏感

Short hairpin RNA expression reversing MDR1 gene-dependent multidrug resistance of human breast cancer cell line (MCF-7/AdrR)

Objective: To reverse multidrug resistance (MDR) of human breast cancer cell line (MCF-7/AdrR) to adriamycin (ADM) with short hairpin RNA (shRNA) expression vectors. Methods: Two shRNA expression vectors harboring MDR1 gene 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 blotting. The apoptosis and sensibility of the breast cancer cells to ADM were evaluated by flow cytometry and MTT assays, respectively. Cellular daunorubicin accumulation was assayed by laser scanning confocal microscope (LSCM).Results: RT-PCR showed that MDR1 mRNA expression was significantly reduced to 37.6 % (P<0.05) and 28.0% (P<0.01) in MCF-7/AdrA cells stably transfected with pSilencer TM 3.1-H1 neo MDR1-A and MDR1-B shRNA, respectively. Western blotting showed that P-gp expression was inhibited significantly and specifically. Resistance against ADM was decreased from 162-fold to 108-fold (P<0.05 ) and 50-fold (P<0.01 ). Furthermore, shRNA vectors significantly enhanced the cellular daunorubicin accumulation. MDR1 shRNA vectors combined with ADM significantly induced the apoptosis of MCF-7/AdrR cells. Conclusion: shRNA vectors can effectively reverse MDR and can restore the sensitivity of drug-resistance cancer cells to conventional chemotherapeutic agents.