An integrative approach to the identification of TS-miRNAs using function- and expression-based screening in HCC cell lines.

<p><b>A</b>, Strategy for the identification of TS- miRNAs by function- and expression-based screening in 6 HCC cell lines. <b>B</b>, Results of function-based screening in 6 HCC cell lines. The numbers of viable cells 4–5 days after transfection with 5 nM of 470 dsRNAs mimicking mature miRNAs loaded on Pre-miR™ miRNA Precursor Library-Human V3 (Ambion) or control non-specific dsRNA were evaluated by the WST-8 assay in duplicate. Relative <i>in vitro</i> cell growth ratios in these cell lines were calculated by normalization of each result to the cell numbers in control cells transfected with non-specific miRNA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060155#pone.0060155.s012" target="_blank">Dataset S1</a>). The lower solid arrow indicates the 470 miRNAs examined. Closed arrows in each graph indicate candidate miRNAs with marked growth inhibitory effects (growth ratio <0.8) compared with the control counterpart in our function-based screening. The arrowhead indicates the results for seven candidate miRNAs conclusively selected in the function- and expression-based screening (see Fig. 1D). <b>C</b>, Summary of expression profiles of 45 candidate miRNAs, expression levels of which were downregulated (>2-fold) in all 6 cell lines as compared with those of two normal liver tissues (C20 and C40, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060155#pone.0060155.s013" target="_blank">Dataset S2</a>). Stars indicate the results of the same seven candidate miRNAs as in Fig. 1B. <b>D</b>, Seven candidates for tumor-suppressive miRNAs involved in HCC pathogenesis identified through function- and expression-based screening approach. MiRNAs frequently downregulated (>50%) in tumors compared with their paired non-tumorous liver tissues in clinical HCC cases are in bold face (^a). Expression level of mature form for each miRNA was evaluated inTaqMan® MicroRNA Assays (Applied Biosystems) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060155#s4" target="_blank">Materials and Methods</a> (^b).