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