FIG. 9.

Downregulation of lin-28 gene expression by production of miR-125b in undifferentiated P19 cells. Undifferentiated P19 cells were transfected with a plasmid (pSH-MIR125b, 1.5 μg) encoding miR-125b or with a variant thereof (pSHAG-1, 1.5 μg) lacking the miR-125b gene segment (Δ), and after two days, RNA and protein extracts were prepared. Alternatively, untransfected P19 cells (−) were treated with retinoic acid for 4 days, and RNA was extracted 1 day later. Equal amounts of each RNA or protein sample were analyzed by blotting to detect miR-125b, Lin-28 protein, and lin-28 mRNA. tRNA, 18S rRNA, and actin served as internal standards. The increased production of miR-125b in undifferentiated P19 cells that had been transfected with pSH-MIR125b reduced the abundance of the Lin-28 protein and lin-28 mRNA to approximately one-quarter (26% ± 2%) and one-half (52% ± 2%) of their respective concentrations in control cells (Δ).