Direct target genes of miR-126. (A) ERRFI1 and FZD7 3′UTR and the wild-type (_WT) and mutant (_Mut) sequences of mature miR-126 (left), and luciferase reporter and mutagenesis assay results (right). Whereas wild-type miR-126 significantly inhibits luciferase activity of the reporter plasmid carrying the 3′UTR of ERRFI1and FZD7, mutation in the miR-126 seed sequence rescues the inhibitory effect. (B) Western blot analysis of Errfi1, Fzd7, and Spred1 levels in AE9a, AE9a+miR-126, and miR-126KO+AE9a AML cells collected from quaternary BMT recipients (2 samples per group). β-Actin was used as an endogenous control. (C) Colony-forming/replating assays with cotransduction of MSCVneo-AE9a together with MSCV-PIG (Control), MSCV-PIG-Errfi1, or MSCV-PIG-Spred1 into normal mouse BM progenitor cells (left), or with transduction of MSCV-PIG (Control), MSCV-PIG-Errfi1, or MSCV-PIG-Spred1 into AE9a+miR-126 BM leukemia cells collected from tertiary BMT recipients (right). (D) Western blot analysis of phosphorylated (P)-MEK1 or P-ERK in human 293T cells transfected with MSCV-PIG (Control) or MSCV-PIG-miR-126 (miR-126) 48 hours posttransfection. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. (E) Colony-forming/replating assays with transduction of MSCV-PIG (Control) and MSCV-PIG-Fzd7 (Fzd7) into AE9a BM leukemia cells collected from tertiary BMT recipients. (F) Correlation between the expression levels of miR-126 and ERRFI1, SPRED1, or FZD7 in the set of 29 AML patients carrying t(8;21). All expression data were log2 transformed and mean centered. The correlation coefficient (r) and P values were detected by Pearson correlation, and the correlation regression lines were drawn with the linear regression algorithm. (G) Comparison of the OS in t(8;21) AML patients with higher or lower levels of ERRFI1, SPRED1, or FZD7 expression. Kaplan-Meier survival curves and P values (log-rank test) are shown.