Forced expression of miR-181a/miR-181b represses endogenous expression of the 4 homeobox target genes, and PBX3 is a direct target of miR-181a/miR-181b. (A-B) Quantitative PCR analyses of effects of the ectopic expression of miR-181a or miR-181b on the expression of the 4 homeobox genes (ie, HOXA7, HOXA9, HOXA11, and PBX3) in human MLL-rearranged leukemic cells (A, using MONOMAC-6 cells as a representative) or in MLL-fusion-mediated mouse primary leukemic BM cells (B). Data are mean ± SD. *P < .05. (C) Luciferase reporter and mutagenesis assays. Left panel: Predicted miR-181a/miR-181b target sites and corresponding mutants in the 3′-UTR of PBX3. Right panel: Forced expression of miR-181a or miR-181b can significantly repress luciferase activity of the reporter gene bearing 3′-UTR of PBX3 in human 293T cells, whereas mutation at the putative target site of miR-181 in the 3′-UTR can abrogate the inhibition. The normalized luciferase activities represent the firefly: β-Galactosidase ratios normalized to the control sample. Error bars present SD obtained from 3 independent experiments. (D) Quantitative PCR (left panel) and Western blot (right panel) assays of the effect of ectopic expression of miR-181a/mIR-191b on the endogenous expression of PBX3. MONOMAC-6 cells served as a representative.