MiR-663 is inactivated by promoter hypermethylation in pediatric AML. (A) MSP analysis of the methylation status of miR-663 in leukemia primary cells 72 h after treatment with 5-Aza, or the same volume of DMSO, shows that the methylation status of the miR-663 promoter is altered in the three primary AML samples. M and U represent MSP results using primer sets for methylated and unmethylated miR-663 genes, respectively. (B) The transcript level of miR-663 is upregulated in primary AML cells treated with 5-Aza compared to DMSO control samples: 2.44-fold in AML10# (5-Aza: 15.9 vs. DMSO: 6.54; P = 0.02); 2.6-fold in AML11# (5-Aza: 35.99 vs. DMSO: 13.9; P = 0.04); and 22.2-fold in AML18# (5-Aza: 11.9 vs. DMSO: 0.54; P < 0.01). (C) Real-time analysis of miR-663 transcript expression in pediatric AML patients shows that patients with miR-663 methylation (n = 29) and those without miR-663 methylation (n = 41) both have significantly lower miR-663 transcript expression levels compared to controls. (D) The transcript level of the miR-663 gene in 70 AML patients is 2.67 ± 3.55 compared to 14.06 ± 9.81 in 30 control samples (P < 0.001).