Fig. 5.

PBX3 and MEIS1 are direct target genes of miR-495. (A) Ectopic expression of miR-495 significantly (P < 0.05) represses endogenous expression of PBX3 and MEIS1 in MLL-rearranged AML cells. The cells were transfected with MSCV-PIG (control) or MSCV-PIG-miR-495, and then the effect of miR-495 overexpression was analyzed 48 h after transfection at both mRNA (Left; detected by qPCR, and the GAPDH expression level was used for normalization) and the protein (Right; detected by Western blot) levels. (B) Inhibitory effect of miR-495 on the endogenous expression of Pbx3 and Meis1 in BM cells of the BM transplantation recipient mice shown in Fig. 3A. Gene levels were normalized to the level of endogenous Gapdh. (C and D) miR-495 directly targets PBX3 (C) and MEIS1 (D) as detected by luciferase reporter and mutagenesis assays. In HEK293T cells, plasmids encoding the wild-type 3′ UTR of PBX3 or MEIS1 (namely, PBX3/MEIS1-3′UTR) or the mutant 3′ UTR in which the predicted miR-495 binding site was mutated (namely, PBX3/MEIS1-3′UTRmut), together with MSCV-PIG or MSCV-PIG-miR-495, were cotransfected with β-gal reporter control vector. Luciferase reporter assays were done 48 h after transfection. Forced expression of miR-495 could significantly repress luciferase activity of the reporter gene bearing the 3′ UTR of PBX3 or MEIS1 in human 293T cells, whereas mutation at the predicted target site in the 3′ UTR abrogated the repression. The normalized luciferase activities represent the firefly: β-gal ratios normalized to the control sample. Error bars present SD obtained from three independent experiments. *P < 0.05; **P < 0.01, two-tailed t test.