DLD-1 cells transfected with 50 nM miR-145 duplex exhibit a reduced cell proliferation as measured by crystal violet growth assay. Data are shown as the mean ± S.D. of four replicates.

A, The percentages of genes in the up, down (dn) and no-change (nc) sets with seeds sites in their 3′UTRs. Only those 6mers which are not part of a 7mer or 8mer are reported as 6mers and only those 7mers that are not contained within a 8mer are counted as 7mers. Mean log fold-changes were 0.36, 0.02 and −0.40 for the up, down and no-change sets, respectively. The p-values are calculated testing the null hypothesis that the proportion of genes with seed sites are the same for the down-regulated and the up-regulated genes (dn vs. up) or the down-regulated genes compared to the no-change genes (dn vs. nc). P-values for 7mer seed site enrichment were 1.7·10⁻³³ (dn vs. up) and 2.9·10⁻¹¹ (dn vs. nc). P-values for 7mer-1A seed site enrichment were 3.7·10⁻¹⁸ (dn vs. up) and 7.8·10⁻⁷ (dn vs. nc). B, Unbiased word analysis showing the running sum of the overrepresentation score for the miR-145 7mer seed site in the ranked list of 3′UTR sequences (black line) compared to permutations of the ranked gene list (red lines). Down- and up-regulated genes (defined as genes with FC<−1.1 or FC>1.1) in the ranked gene list are indicated in the graph. C, Quantitative RT-PCR validation of the microarray data. Cells were transfected with 50 nM miR-145 duplex or mock transfected and total RNA harvested 24 hours post-transfection. The 3′UTRs of IQGAP1 and STAT1 do not contain any miR-145 seed matches, but both genes contain a 7mer seed match in their coding region. All other miR-145 responsive genes contain at least one 7mer seed site in their 3′UTR. The expression level of each transcript is shown relative to the level in mock transfected cells. Data are shown as the mean ± S.D. of three replicates.

A, Sequence alignment of the miR-145 seed region and mRNA targets. The position coordinates are indicated for the transcript isoforms listed below. YES1 3′UTR: ENSG00000176105:ENST0000035983 STAT1 cDNA: ENSG00000115415:ENST00000361099. B, Firefly luciferase assay with pGL3+ constructs holding a 3′UTR fragment of YES or a cDNA fragment of STAT1, downstream to the firefly luciferase gene. Cells were co-transfected with firefly luciferase reporters along with a Renilla luciferase transfection control plasmid either alone or with 50 nM miR-145 duplex and 50 nM AllStar duplex as a negative control. In pGL3+ mut vectors two base pairs in the miR-145 seed site have been mutated. Luminescence was measured 24 hours post-transfection and the ratio of the firefly to the Renilla activity is shown relative to transfection without RNA duplex and the empty vector. Data are shown as the mean ± S.D. of four replicates. *, p<0.05 using a two-tailed t-test, ***, p<0.01 using a two-tailed t-test. C and D, Western blot analysis of DLD-1 and HCT116 cells transfected with miR-145 duplexes or mock transfected cells blotted for YES (C) and STAT1 (D). Vinculin or tubulin were used as loading controls. The bands were quantified relative to the appropriate loading controls using the TotalLab software and are shown relative to the protein level in mock transfected cells. The data shown are representative of two experiments.