Knockdown Of MRP4by Lentivirus-mediated SiRNA Improves Sensitivity To Adriamycin In Adriamycin-resistant Acute Myeloid Leukemia Cells

Objective:Acute myeloid leukemia (AML) is a clonal disorder, characterized by the accumulation of an immature cell population in the bone marrow and/or peripheral blood. Despite improvements accomplished in the last30years on the use of combination various cytarabine and anthracyclines agents, the overall prognosis for AML remains poor. A major issue in the treatment of AML is multidrug resistance. Therefore, reversion multi-drug resistance of leukemia cells is important to effectively cure AML patients. Multidrug resistance-associated protein4(MRP4) is a member of the C subfamily of ATP-binding cassette transporters. Studies have shown that high levels of expression of MRP4, but not of any of the other MRP gene family members, were significantly associated with poor outcome in many cancers. However, the importance of MRP4gene in acute myeloid leukemia is unknown. Bone marrow of AML,the adriamycin-resistant acute myeloid leukemia cell lines K562/ADR and parental cell lines K562were used to investigate that knockdown of MRP4using RNAi whether could reverse resistance and sensitize K562/ADR cells to adriamycin.Methods:Bone marrow samples from75patients with de novo AML and23patients with relapse were examined by RT-PCR and bone marrow samples from20normal donors were used as control. The expression of MRP4protein in the K562and K562/ADR cells were detected by Western blot analysis. Five lentivirus-mediated short hairpin RNAs (lv-shRNAs-MRP4) were designed for triggering of the gene silencing RNA interference (RNAi) pathway. The infection efficiency of lentivirus-mediated siRNA into K562/ADR cells expressing GFP protein were observed using fluorescence microscopy. MRP4gene expression in transfected K562/ADR cells were evaluated with real-time PCR and Western blot analysis. MTS assay was used to measure the cell viability and Flow cytometric assay for apoptosis.Results:MRP4mRNA in de novo and relapse AML were significantly higher than that in normal controls (p<0.05). MRP4mRNA in relapse AML was significantly higher than that in de novo AML simultaneously (p<0.05). Expression of MRP4mRNA was highter in patients with high white blood cell count and intermediate/poor karyotype than that with low white blood cell count and good karyotype. Survival in patients with high MRP4expression was significantly shorter than that in patients with low MRP4expression. The MRP4protein expression of K562/ADR cells was incresed than that of K562cells. The transfection efficiency into K562/ADR cells was over80percents. The gene silencing efficacy of lv-shRNA1-MRP4was stronger than the others. The gene silencing efficacy of lv-shRNA1-MRP4was over80percents and stronger than that of the others (p<0.05). The expression of MRP4mRNA and protein in lv-shRNA1-MRP4cells was lower than that of control cells (p<0.05). Combined treatment with lv-shRNA1-MRP4and adriamycin simultaneously was able to decrease the cell growth and increase cell apoptosis compared to single treatment.Conclusion:The expression of MRP4in AML patients, suggesting that the protein may have relationships with pathogenesis and prognosis of the disease. Moreover, it indicate that MRP4is involved in the drug resistance mechanisms of K562/ADR cells and lentivirus-mediated knockdown of MRP4may enhance sensitivity to adriamycin in K562/ADR cells.