FIG. 7.

Overexpression of miR-181a in THP1 cells inhibits the translation of endogenous p27 mRNA. (A) p27 protein levels in HL60 and THP1 cells measured by Western blotting. β-Actin was used as a loading control. (B) Analysis of miR-181a expression. THP1 cells were infected with either empty or primary miR-181a (pri-miR-181a)-expressing retroviruses and selected in neomycin-containing medium. Total RNA was isolated, and the amounts of miR-181a and miR-221 were analyzed by Northern blotting. Untreated HL60 cells were carried as a control. miRNA levels were normalized according to U6 RNA levels, and the resulting values are indicated relative to those found in control THP1 cells. (C) p27 expression decreases in THP1 cells that overexpress miR-181a. Protein levels were analyzed by Western blotting using eIF4E as a loading control. A nonspecific band is also shown as a reference. The p27 values are indicated after correction for eIF4E. (D) Analysis of endogenous p27 mRNA levels in control or miR-181a-overexpressing cells, as determined by qRT-PCR. The average of triplicate RT-PCRs for one biological sample is shown.