FIGURE 4.

p16 positively regulates miR-141 and miR-146b-5p at the transcriptional level. A, Cells were treated with actinomycin D for the indicated periods of time. Total RNA was extracted, and the remaining levels of miR-141 and miR-146b-5p were assessed by RT-PCR following electrophoresis on 2% ethidium bromide-stained agarose gels. B, schematic diagrams of miR-141 and miR-146b-5p genomic loci and their putative promoters. Chr, chromosome; EST, expressed sequence tag; TSS, transcription start site. C, miR-141 and miR-146b-5p promoters were inserted into SmaI/HindIII sites in the pGL3 luciferase reporter plasmid. The pGL3 vector was used to transfect U2OS and EH1 cells together with Renilla luciferase phRLTK vector to normalize for variation in transfection efficiency, and the firefly (FL) and Renilla (RL) luciferase activities were measured 24 h post-transfection using bio-imaging apparatus and software (BD Biosciences). Histogram shows the activity of luciferase under miR-141 and miR-146b-5p promoters. Data are presented as means ± S.E. of three independent experiments and each is measured in triplicate (*, p < 0.05).