miR-181a regulates the level of ETV6/RUNX1.

Overexpression of miR-181a in REH cells was performed by transfection with precursor miRNA. A final 50 nM concentration of nontargeting-miR (NC) or pre-mir-181a (181a) were transfected twice and cells were harvested after 48 hours of the second transfection for further examination. (A) miR-181a level was detected by TaqMan qRT-PCR. (B) Regulation of ETV6/RUNX1 by miR-181a was confirmed by Western blotting with RUNX1-specific (E/R) antibody. Relative expression as determined by densitometry is indicated below the blots. (C) The putative miR-181a binding site in the RUNX1 3' UTR. nt, nucleotides. (D) The last 678 bp of the human RUNX1 3' UTR containing normal (WT) or mutated (mut) miR-181a targeting sequences were cloned downstream of a pGL3-luciferase vector and transfected into 293FT cells with expression vectors for miR-181a (181a) or negative control shRNA (NC). All experiment was conducted in triplicate. Bars represent the mean ± SD of three independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 (ANOVA).