MiR-24 reduces activin A-induced hemoglobin accumulation in K562 cells. (A) The expression of miR-24 in K562 cells and HPCs was assessed by stem-loop RT-PCR with U6 as loading control. Murine lung miR-24 was a positive control. K562 cells were cotransfected with CAGA12-luciferase along with miRNA vectors (B) or anti-miRNA oligoribonucleotides (C) as indicated. After treated with activin A (25 ng/mL) for 16 hours, the cells were harvested for luciferase assay. Reporter assay was performed in triplicate, and the data represent the mean plus or minus SD of 3 independent experiments after normalized to R. reniformis activity. K562 cells were transfected with the indicated constructs or anti-miRNA oligoribonucleotides. The transfected cells were placed in the absence or presence 25 ng/mL activin A for 3 days and processed for benzidine staining. The results are expressed as percentage of benzidine-positive (blue) cells compared with the total cell number. The values represent the means plus or minus SD of 3 separate experiments. A representative field (original magnification 100×) (D) and quantitation of 3 independent experiments (E,F) were shown. The asterisk indicates a significant difference between miR-24 and control vector or between anti-24 and anti-24M oligoribonucleotides (P < .05). (G) K562 cells were cotransfected with indicated miRNA plasmids and treated with activin A for 1 hour before the cells were harvested for Western blotting to reveal the protein levels of hALK4 (top panel), phospho-Smad2 (3rd panel), or total Smad2 (4th panel).
