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. 2015 Sep 14;10(9):e0137887. doi: 10.1371/journal.pone.0137887

Fig 1. Verification of miR-19a candidate target genes by luciferase assays.

Fig 1

(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 XbaI restriction site of the pTK-hRG vector. Sense (upper) and antisense (lower) 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 XbaI 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. *, p < 0.05 and **, p < 0.005 using two-tailed t-tests.