<i>miR-655</i> Is an EMT-Suppressive MicroRNA Targeting <i>ZEB1</i> and <i>TGFBR2</i>

<div><p>Recently, the epithelial-to-mesenchymal transition (EMT) has been demonstrated to contribute to normal and disease processes including cancer progression. To explore EMT-suppressive microRNAs (miRNAs), we established a cell-based reporter system using a stable clone derived from a pancreatic cancer cell line, Panc1, transfected with a reporter construct containing a promoter sequence of <i>CDH1/E-cadherin</i> in the 5′ upstream region of the <i>ZsGreen1</i> reporter gene. Then, we performed function-based screening with 470 synthetic double-stranded RNAs (dsRNAs) mimicking human mature miRNAs using the system and identified <i>miR-655</i> as a novel EMT-suppressive miRNA. Overexpression of <i>miR-655</i> not only induced the upregulation of E-cadherin and downregulation of typical EMT-inducers but also suppressed migration and invasion of mesenchymal-like cancer cells accompanied by a morphological shift toward the epithelial phenotype. In addition, we found a significant correlation between <i>miR-655</i> expression and a better prognosis in esophageal squamous cell carcinoma (ESCC). Moreover, <i>ZEB1</i> and <i>TGFBR2</i>, which are essential components of the TGF-b signaling pathway, were identified as direct targets of <i>miR-655</i>, suggesting that the activation of the TGF-b-ZEB1-E-cadherin axis by aberrant downregulation of <i>miR-655</i> may accelerate cancer progression.</p></div

EMT-suppressive effects of <i>miR-655</i> on mesenchymal-like cancer cells having phenotypic plasticity at EMT/MET.

<p><b><i>A</i></b>, TaqMan real-time RT-PCR analysis of <i>CDH1/E-cadherin</i> and <i>miR-655</i> in a panel of 23 pancreatic cancer cell lines and a breast cancer cell line, MDA-MB-231. Relative expression levels of transcripts of <i>CDH1/E-cadherin</i> and <i>miR-655</i> were quantified in comparison to <i>GAPDH</i> and <i>RNU6B</i>, respectively, to normalize the initial input of total RNA. Bar graphs show the ratio of the expression level in these cell lines to that in normal pancreas (Ambion). <b><i>B</i></b>, Representative results of phase contrast images (<b><i>Upper</i></b>) and <i>CDH1/E-cadherin</i> protein expression level detected by immunofluorescence staining (<b><i>Lower</i></b>) in Panc1, KP1N, KP4-4 and MDA-MB-231 cells 96 hours after transfection with 10 nM of ds-<i>NC</i> or dsRNA mimicking <i>miR-655</i> (ds-<i>miR-655</i>) (Ambion). <b><i>C</i></b>, TaqMan real-time RT-PCR analysis (<b><i>Upper</i></b>) and Western blot (<b><i>Lower</i></b>) analysis of mRNA and protein levels of <i>CDH1/E-cadherin</i>, respectively, in Panc1, KP1N, KP4-4 and MDA-MB-231 cells 96 hours after transfection of 10 nM of ds-<i>NC</i> or ds-<i>miR-655</i>. Asterisks (*), statistical analysis with the Mann-Whitney <i>U</i> test. <b><i>D</i></b>, Growth curves in Panc1, KP1N, KP4-4 and MDA-MB-231 cells after transfection of 10 nM of ds-<i>NC</i> or ds-<i>miR-655</i>. Each data point represents the mean of duplicate determinations (bars, SD) in these experiments. Asterisks (*), statistical analysis with the Mann-Whitney <i>U</i> test. <b><i>E</i></b>, Representative phase micrographs of Panc1, KP1N, KP4-4 and MDA-MB-231 cells transiently transfected with 10 nM of ds-<i>NC</i> or ds-<i>miR-655</i> in cell migration and invasion assays <i>in vitro</i> using uncoated and Matrigel-coated transwell-chamber culture systems (Becton Dickinson), respectively. At 48 hours after transfection of dsRNA, cells were transferred into the upper chamber of the transwell (4×10⁴ cells per well). The migrating or invading cells on the lower surface of filters were fixed and stained with the Diff-Quik stain 48 hours after cell transfer. <b><i>F</i></b>, Quantification of the cell migration (<b><i>Left</i></b>) and invasion (<b><i>Right</i></b>) shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062757#pone-0062757-g003" target="_blank">Figure 3B</a>. Bar graphs show the percentage (%) of <i>miR-655</i>-transfectants migrating (<b><i>Left</i></b>) or invading (<b><i>Right</i></b>) through uncoated or Matrigel-coated filters, respectively, relative to control-transfectants. Asterisks (*), statistical analysis with the Mann-Whitney <i>U</i> test.</p

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.</p

Expression analysis of <i>miR-655</i> in primary ESCC and OSCC cases.

<p><b><i>A</i></b>, TaqMan real-time RT-PCR analysis of endogenous <i>miR-655</i> in 22 normal human tissues (Ambion and Clontech). Marked upregulation of <i>miR-655</i> expression (>2-fold increase compared with pancreas) was observed in brain, cervix, esophagus and placenta. <b><i>B</i></b>, Expression profiles of <i>miR-655</i> in a panel of paired tumorous and non-tumorous tissues from primary ESCC and OSCC cases. Bar graphs show the ratio of the expression level in tumors (T) to those in their paired normal mucosae (N). <b><i>C</i></b>, Kaplan-Meier survival curves for high and low <i>miR-655</i> groups based on TaqMan real-time RT-PCR. In univariate analyses of overall and non-recurrent survival with log-rank tests, a high level of <i>miR-655</i> expression was significantly associated with a much better survival rate among patients with ESCC (<i>P</i> = 0.0359, log-rank test).</p

Summary of 17 miRNA genes selected as candidates for EMT-suppressive miRNAs in functional-based screening using a stable Panc1 clone transfected with a reporter construct containing a promoter sequence of <i>CDH1/E-cadherin</i> in the 5′ upstream region of the <i>ZsGreen1</i> reporter gene and Pre-miR™ miRNA Precursor Library - Human V3 (Ambion).

*<p>The ratio of fluorescence intensity of ZsGreen1 (RFI) in cells 4 days after transfection with each dsRNA was normalized to that in control transfectants (Pre-miRTM Negative Control #1, Ambion).</p>**<p>The ratio of growth level (RG) of viable cells assessed by WST8 assay 4 days after transfection with dsRNAs. WST-8 assay was employed to normalize the number of viable cells relative to the control transfectants.</p