siRNA-mediated silencing of ETV6/RUNX1 increases the level of mature miR-181a-1.

(A) Stem-loop sequence of human precursor mir-181a-1. Mature miR-181a (red) and miR181a-1 (blue) are indicated. (B) Validation of miR-181a-1 expression in primary pre-B ALL samples (ETV6/RUNX1-positive, n = 7; ETV6/RUNX1-negative, n = 8) using TaqMan qRT-PCR. ETV6/RUNX1-expressing REH cells (control) were transfected with siRNAs. After 48 hours of transfection with functional siETV6/RUNX1 (siE/R) or nonfunctional siRNA (siRNA-S), (C) ETV6/RUNX1 mRNA was detected by qRT-PCR, and (D) protein was analyzed by Western blotting with anti-RUNX1; anti-β-actin was used as a loading control. Relative expression as determined by densitometry is indicated below the blots. (E) Mature miR-181a-1 was measured by qRT-PCR. Bars represent the mean ± SE of at least three independent experiments. GAPDH and RNU6B were used as calibration controls for mRNA and miRNA expression, respectively. *P ≤ 0.05, **P ≤ 0.01 (ANOVA).