Verification of miR-19a candidate target genes by luciferase assays.

<p>(A) Overview of miR-19a target candidate mRNAs. The miR-19a–binding sites identified using PicTar, TargetScan, or MiRanda software are shown in the 3ʹ-untranslated (UTR) region of miR-19a target candidate mRNAs. (B) Construction of luciferase vectors. The miR-19a–binding sites of the candidate genes and negative control sequences (Negative Ctrl) were cloned downstream of the luciferase ORF at the <i>Xba</i>I restriction site of the pTK-hRG vector. Sense (<i>upper</i>) and antisense (<i>lower</i>) strands of complementary sequences indicate the miRNA target site of the mRNA 3ʹ-UTR and miR-19a sequences, respectively. Underlines indicate miR-19a seed sequences. The negative control plasmid has mismatches in the center of miR-19a seed sequences. (C) The luciferase activity of constructs with miR-19a–binding sites was compared with that of the empty vector, which had no insert at the <i>Xba</i>I site (No-Insert Ctrl), and was statistically analyzed. (D) Luciferase plasmids with the miR-19a–binding site and negative control plasmid were transfected with anti-miR-19a-LNA or control-LNA (Ctrl). The luciferase activity of cells treated with anti-miR-19a-LNA was compared with that of cells treated with the control-LNA. *, <i>p</i> < 0.05 and **, <i>p</i> < 0.005 using two-tailed <i>t</i>-tests.