Function-based screening of EMT-suppressive miRNAs using reporter system for investigating <i>CDH1/E-cadherin</i>-promoter activity in Panc1 cells.

<p><b><i>A</i></b>, Map of the promoter region of the <i>CDH1/E-cadherin</i> gene. To construct a reporter plasmid, 1,058 bp promoter sequences indicated by the closed arrow in this map was introduced into a promoterless pZsGreen1-1 vector with the <i>ZsGreen1</i> gene as a reporter gene. A cell-based reporter system was established by isolation of a stable clone with the limiting dilution method after transfection of the construct into Panc1 cells. <b><i>B</i></b>, Confirmation of the expression of the ZsGreen1 protein in the cell-based reporter system following transfection of <i>miR-200a</i> or <i>-200b</i>. A stable cell clone with the reporter plasmid was evaluated 96 hours after transient transfection of 10 nM of dsRNA mimicking <i>miR-200a</i> or <i>-200b,</i> or control non-specific miRNA (ds-<i>miR-200a</i>, ds-<i>miR-200b</i> or ds-<i>NC</i>) (Ambion). <b><i>Upper</i></b><b>,</b> Detection of ZsGreen1 in these transfectants using fluorescence micrographs. <b><i>Lower</i></b><b>,</b> Quantification of fluorescence intensity in these transfectants (<b><i>Left</i></b>). Results of the TaqMan real-time RT-PCR analysis (<b><i>Middle</i></b>) and Western blot analysis (<b><i>Right</i></b>) for expression of the <i>CDH1/E-cadherin</i> transcript and protein, respectively, in these transfectants. <b><i>C</i></b>, Results of the function-based screening of EMT-suppressive miRNAs in a cell-based reporter system using Pre-miR™ miRNA Precursor Library-Human V3 (Ambion) containing 470 dsRNAs mimicking human mature miRNAs. The fluorescence intensity of ZsGreen1 was evaluated by fluorescence microplate reader in duplicate. The relative fluorescence intensity in each transfectant was calculated by normalization of each result to the fluorescence intensity in control cells transfected with non-specific miRNA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062757#pone-0062757-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062757#pone.0062757.s011" target="_blank">Table S2</a>). The lower closed arrow indicates the 470 miRNAs examined. <b><i>D</i></b>, Western blot analysis of E-cadherin protein levels in parental Panc1 cells 96 hours after transient transfection with 10 nM of ds-<i>NC</i> or 10 nM of ds-miRNAs mimicking <i>miR-96-5p, -132-3p, -183-5p, -139-5p, -217, -520d-3p, -526b-3p, -629-3p, -655</i> and <i>-200b-3p</i>. Because <i>miR-200b</i> has already been confirmed to induce expression of the <i>CDH1/E-cadherin</i> transcript and protein in this study (Fig. 1B) and multiple previous studies, ds-<i>miR-200b</i> was used as a positive control in this analysis.