Stable expression of miR-17 deregulates the same core RUNX1-miR221-KIT axis affected by CBF-AML fusion proteins. (A) Stable U937 clones ectopically expressing miR-17 (U937miR-17) or a control scrambled sequence (U937Scram) were developed by transfection with a construct co-expressing GFP and the miRNA precursor of interest (left). Cytofluorimetric analysis shows GFP expression in representative U937miR-17 and U937Scram clones (right). (B) Stable ectopic expression of miR-17 leads to RUNX1 downregulation, as shown by decreased luciferase activity (bottom) in a representative U937miR-17 clone transfected with a reporter plasmid carrying luciferase upstream of RUNX1-3′UTR (top). (C) Ectopic expression of miR-17 increases both the number of KIT-positive cells (p < 0.05) (left) and cell proliferation (right). (D) Like RUNX1-MTG8 and CBFB-MYH11, miR-17 downregulates miR-221 expression (left). Consistently, both CBF-AML and non-CBF-AML display miR-221 downregulation (right). (E) U937 cells transiently transfected with a synthetic anti-miR inhibiting endogenous miR-221 activity display a significant (p <0.01) increase of KIT-positive cells (left) and increased proliferation (right) relative to U937 transiently transfected with a control anti-miR scrambled sequence. (F) Stable ectopic expression of wild type KIT in U937 cells (U937KIT) (left) is sufficient to promote proliferation (right). (G) These results indicate that miR-17, by affecting the RUNX1-miR-221 mechanism, leads to KIT upregulation. Shown here one representative clone out of 3 clones stably expressing RUNX1-MTG8, CBFB-MYH11, or miR-17.