Inhibition Of Mdr1 Gene Expression By MicroRNA-mediated RNA Interference In Tumor Cells

Objective To investigate the effect of microRNA-mediated RNA interference(RNAi) on the expression of mdr1 gene and the reversal of multidrug resistance(MDR) of MCF7/ADR human breast cancer cell line.Methods Four pairs of single-stranded oligo encoding mdrl pre-miRNA sequence were designed and synthesized.After annealing ds oligo were inserted into polⅡmiRNA expression vector,which is based on the routine miR-155 sequence,to construct recombinant vectors.Then four recombinant vectors and one negative-control vector (mdr1-miRNA-Neg) were transfected into MCF7/ADR cells by liposome.The expression of mdr1 mRNA and P-gp protein were detected by real-time RT-PCR and western blot, respectively.The sensitivity of the cells to adriamycin was detected by MTT assay.Results Recombinant vectors were successfully constructed and transfected.The results showed that two of four recombinant vectors(mdr1-miRNA-1 and mdrl-miRNA-2) could effectively cleave mdrl mRNA and the expression of P-gp protein was also significantly decreased.The mRNA expression in mdrl-miRNA-1 and mdrl-miRNA-2 transfected MCF7/ADR ceils was significantly reduced to 42.60%(P＜0.01) and 49.23%(P＜0.01),respectively.Western blot analysis demonstrated P-gp expression was significantly reduced to 51.61%(P＜0.05) and 52.18%(P＜0.05),respectively.The resistance index(RI) of MCF7/ADR cells transfected with mdr1-miRNA-1 vector was decreased to 39.85%, from 138-fold to 55-fold(P＜0.01).Mdr1-miRNA-1 vector decreased the resistance index to 54.49%,from 138-fold to 75-fold(P＜0.01).The difference among mdrl-miRNA-3, mdrl-miRNA-4,mdrl-miRNA-Neg and blank control group had no statistical significance(P＞0.05).Conclusion MicroRNA-mediated RNA interference can effectively inhibit the expression of mdr1 gene by mRNA cleavage and restore the sensitivity of MCF7/ADR cells to adriamycin.These results may provide a new strategy for the reversal of multidrug resistance of tumor cells and lay a foundation for the application of microRNA to RNAi and gene therapy.