RNAi-Mediated Gene Silencing Of Breast Cancer Resistance Protein Reverse Drug-Resistance Of MCF-7/ADR

Objective :Multidrug resistannce is the important mechanism of tumor drug resistance,and the optinal reason leading to chemotherapy failture .MDR including many protein,such as : P-glyeoprotein(P-gp),multidrug resistance-associated protein (MRP1～7) and breast cancer resistance protein (BCRP). AS drug pump ,they could reduce drug concentration in cell and result in drug resistance.The expression of BCRP in MCF-7/ADR results in drug resistance directly,therefore blocking the expression of BCRP could reverse the drug resistance of MCF-7/ADR.RNA interference is post transcriptional gene silencing by double-stranded RNA(dsRNA).In recent years ,the RNA interference technique has become a new and hot research field for gene therapy because it can block the target gene in mammalian cells.So ,we can design short interfering RNA(SiRNA) for BCRP mRNA,block the expression of target gene ,reverse the drug resistance and could be a new therapy way for tumor.Methods: Design ,filtrate and synthesize three SiRNA sequence for BCRPmRNA (BCRP1, BCRP2, BCRP4), and construct pGenesil -BCRP- SiRNA-EGFP expression vector.Identify and validate the validity of sequence. pGenesil-BCRP-SiRNA-EGFP expression vector was transfected into MCF-7/ADR cell line by liposome .The positive transfected cell clones were screened with G418.Expression of BCRP mRNA and protein in MCF-7/ADR cells after gene transfer was detected by semi-quantitative reverse transcription polymerse chain reaction and Western Blot assay, respectively.The changes of IC50 were analysised by MTT assay.Finally, we could filtrate the best interfering sequence .Results: pGenesil-BCRP-SiRNA-EGFP expression vector was successfully constructed .The expressions of BCRP mRNA and protein were decreased differently ,BCRP1 has the best interfering effects.The inhibitive ratio of BCRP1 was 30.3% and 65.6% respectively (p<0.05).After transfected, the IC50 of each interfering group has changed differently, BCRP1 group has decreased markedly(p<0.05).Conclusion: pGenesil-SiRNA-EGFP expression vector was successfully constructed .The expressions of BCRP mRNA and protein were decreased differently; BCRP1 has the best interfering effects. The drug resistance was reversed different degree.These results suggest that a strategy based on SiRNA targeting of BCRP may build the foundation to the clinical chemotherapy for breast cancer.