The aim of this study was to investigate the effect of a eukaryotic expression vector expressing hsa-miR-203 on the sensitivity of K562 leukemia cells to arsenic trioxide (ATO) and the possible mechanism of action. The eukaryotic expression vector expressing the hsa-miR-203 plasmid (PmiR-203) was transfected into K562 cells using Lipofectamine 2000. bcr/abl 3' untranslated region (UTR) and bcr/abl mutated 3'UTR dual luciferase report vectors (psi-CHECK-2) were used to validate the regulation of bcr/abl by miR-203. The inhibitory effects of ATO and PmiR-203, used singly or in combination, on cell proliferation were detected by MTT assay. Apoptosis of the K562 cells was detected by flow cytometry using double-staining with Annexin V and propidium iodide (PI). The activities of caspase-3 and caspase-9 were detected by a colorimetric method and the cytochrome c protein levels were detected by western blotting. When used in combination with PmiR-203, the IC50 of ATO was reduced from 6.49 to 2.45 μg/ml and the sensitivity of cells to ATO increased 2.64-fold. In addition, PmiR-203 and ATO caused growth inhibition, apoptosis and G1-phase arrest in K562 cells. Furthermore, PmiR-203 significantly promoted ATO-mediated growth inhibition and apoptosis, affecting the G1 phase. JC-1 fluorescent staining revealed that the membrane potential of the mitochondria had changed. The activities of caspase-3 and caspase-9 increased, the expression levels of cytochrome c were upregulated and the expression level of bcr/abl mRNA was significantly suppressed. Furthermore, the dual-luciferase reporter vector, containing tandem miR-203 binding sites from the bcr/abl 3'UTR, demonstrated that bcr/abl was directly regulated by miR-203. PmiR-203 sensitized K562 leukemia cells to ATO by inducing apoptosis and downregulating bcr/ abl gene levels. The induction of apoptosis may occur through the mitochondrial pathway. The combination of ATO and PmiR-203 presents therapeutic potential for chronic myelogenous leukemia.
Keywords: apoptosis; arsenic trioxide; chronic myelogenous leukemia; eukaryotic expression vector; hsa-miR-203.