by miR-139-5p. A. Potential wild-type miR-139-5p target site (top) and a mutant with seed mismatches indicated by asterisks (bottom) in the ZNF265 3′ UTR. B. Relative luciferase activity from the Renilla-luciferase reporter constructs containing either the wild-type (black bars) or mutated (gray bars) ZNF265 3′ UTR in parental and ORF2-expressing PK15 cells (top left), and in parental PK15 cells with or without (empty vector) miR-139-5p overexpression (top right). The level of mature miR-139-5p in each sample was also determined by splinted ligation assay (bottom). The 5.8S rRNA served as a loading control. Note that miR-139-5p is down-regulated in ORF2-expressing cells compared with parental cells (bottom left). For quantification, Renilla-luciferase activity was normalized to firefly-luciferase activity. The normalized activity of Renilla luciferase in parental cells or the same cells transfected with the empty vector was set to 1. Data are presented as the mean ± SEM from three independent experiments. NS, no significance; **P < 0.01 by Student’s t-test. Abbreviations: RL, Renilla luciferase; FL, firefly luciferase.