摘要
Chemotherapy remains the standard treatment for acute myeloid leukemia;however,the emergence of drug resistance is a major hurdle in the successful treatment of leukemia.The expression of multidrug resistance-associated protein 4(MRP4)induces re- sistance in the adriamycin-resistant acute myeloid leukemia cell line,K562/ADR.The aim of this study was to investigate whether knockdown of MRP4 by lentivirus-mediated siRNA could improve the sensitivity of K562/ADR cells to adriamycin.Five lenti- virus-mediated short hairpin RNAs(lv-shRNAs-MRP4)were designed to trigger the gene silencing RNA interference(RNAi) pathway.The efficiency of lentivirus-mediated siRNA infection into K562/ADR cells was determined using fluorescence mi- croscopy to observe lentivirus-mediated GFP expression.MRP4 expression in infected K562/ADR cells was evaluated by real- time PCR and Western blot analysis.The MTS assay was used to measure cell viability and flow cytometry was used to measure apoptosis.The transfection efficiency of K562/ADR cells was over 80 percent.The gene silencing efficacy of lv-shRNA1-MRP4 was superior to the other constructs.Infection of K562/ADR cells with lv-shRNA1-MRP4 led to strong inhibition of MRP4 mRNA and protein expression.Combined treatment with lv-shRNA1-MRP4 and adriamycin decreased cell growth and increased apoptosis compared to treatment with lv-shRNA1-MRP4 or adriamycin alone.These data indicate that in K562/ADR cells MRP4 is involved in drug resistance mechanisms and that lentivirus-mediated knockdown of MRP4 may enhance sensitivity to adriamycin.
Chemotherapy remains the standard treatment for acute myeloid leukemia; however, the emergence of drug resistance is a major hurdle in the successful treatment of leukemia. The expression of multidrug resistance-associated protein 4 （MRP4） induces resistance in the adriamycin-resistant acute myeloid leukemia cell line, K562/ADR. The aim of this study was to investigate whether knockdown of MRP4 by lentivirus-mediated siRNA could improve the sensitivity of K562/ADR cells to adriamycin. Five lentivirus-mediated short hairpin RNAs （lv-shRNAs-MRP4） were designed to trigger the gene silencing RNA interference （RNAi） pathway. The efficiency of lentivirus-mediated siRNA infection into K562/ADR cells was determined using fluorescence microscopy to observe lentivirus-mediated GFP expression. MRP4 expression in infected K562/ADR cells was evaluated by realtime PCR and Western blot analysis. The MTS assay was used to measure cell viability and flow cytometry was used to measure apoptosis. The transfection efficiency of K562/ADR cells was over 80 percent. The gene silencing efficacy of lv-shRNA1-MRP4 was superior to the other constructs. Infection of K562/ADR cells with lv-shRNA1-MRP4 led to strong inhibition of MRP4 mRNA and protein expression. Combined treatment with lv-shRNA1-MRP4 and adriamycin decreased cell growth and increased apoptosis compared to treatment with lv-shRNA1-MRP4 or adriamycin alone. These data indicate that in K562/ADR cells MRP4 is involved in drug resistance mechanisms and that lentivirus-mediated knockdown of MRP4 may enhance sensitivity to adriamycin.
基金
supported by the Natural Science Foundation of Gansu Province(20090501)
