Ectopic expression of miR-181a and, particularly, miR-181b exhibits anti–tumor effects in MLL-rearranged AML in vitro and in vivo. *P < .05 (2-tailed t test). (A) Effect of ectopic expression of miR-181a and miR-181b on viability (left panel) or apoptosis (right panel) of MONOMAC-6, THP-1, and KOCL-48 cells, respectively. The controls are cells transfected with empty plasmids. Data are mean ± SD from 3 independent experiments. (B) Cell growth/proliferation analysis of MONOMAC-6 cells transfected with MSCV-PIG (ie, control), MSCV-PIG-miR-181a, or MSCV-PIG-miR-181b. Cell growth is significantly (P < .05) slower in leukemic cells transduced with miR-181a or miR-181b than those transduced with empty vector after 3 days of culture. (C) Colony-forming/replating assay of mouse normal BM progenitor cells transduced with MSCVneo + MSCV-PIG (ie, control), MSCVneo + MSCV-PIG-miR-181a (ie, miR-181a), MSCVneo + MSCV-PIG-miR-181b (ie, miR-181b), MSCVneo-MLL-AF9 + MSCV-PIG (ie, MA9), MSCVneo-MLL-AF9 + MSCV-PIG-miR-181a (ie, MA9 + miR181a), or MSCVneo-MLL-AF9 + MSCV-PIG-miR-181b (ie, MA9 + miR181b). Duplicates were plated for each combination with 1 × 10⁴ cells per dish, and every 7 days the cells were replated for up to 4 passages. Two independent experiments were conducted. Data are mean ± SD. (D) In primary mouse bone marrow transplantation (BMT) assay, both MA9 + miR181a mice (n = 5) and MA9 + miR181b mice (n = 5) developed leukemia slower than MA9 (ie, MLL-AF9 alone) mice (n = 5), although only the difference between MA9 + miR181b and MA9 mice is statistically significant (P = .004; log-rank test). (E) Relative expression of miR-181a or miR-181b expression in MA9, MA9 + miR181a, or MA9 + miR181b leukemic mouse BM cells (samples from 3 mice in each cohort were analyzed). The level in MA9 was set as 1.