Predicted miRNAs and target FZDs.

<p>Predicted miRNAs and target FZDs.</p

Effects of <i>miR-203</i> and <i>miR-338-3p</i> transfection on the migration of h1299 cells.

<p>(A–F) Cell migration was evaluated using wound healing assays, as described in the Materials and Methods. Randomly chosen wound fields were photographed every 8 h for 24 h. (G, H) Wound areas were evaluated using the following formula: wound area (%) = wound area after the indicated period × 100 / initial wound area. (I) Migration ability was evaluated based on the wound area. The experiments were performed in triplicate, and the data were calculated as means ± SDs. The statistical significance of differences was analyzed using the Student’s t-test. *<i>P</i> < 0.01, **<i>P</i> < 0.05.</p

Transfection with miRNA mimics and hairpin inhibitors.

<p>Total RNA was isolated from h1299 cells at 48 h after transfection with miRNA mimics or miRNA hairpin inhibitors, and miRNA levels were then analyzed by real-time RT-PCR. <i>RNU6B</i> was used as an internal reference gene. Experiments were performed in triplicate, and relative miRNA levels (zeo/control inhibitor = 1) were averaged. The asterisks in the figure indicate statistically significant differences (*<i>p</i> < 0.01) between the two values. Data are presented as the means ± SDs.</p

Expression of 11 miRNAs in h1299 cells.

<p>Total RNA was isolated from h1299 cells, and the 11 predicted miRNA levels were analyzed by real-time RT-PCR. <i>RNU6B</i> was used as an internal reference gene. Experiments were performed in triplicate, and relative miRNA levels (zeo = 1) were averaged. Asterisks indicate statistically significant differences (*<i>p</i> < 0.05, **<i>p</i> < 0.01) between the two values. Data are presented as the means ± SDs.</p

Target inhibition assay for determination of the effects of <i>miR-203</i> on <i>FZD2</i> mRNA expression.

<p>An <i>miR-203</i> mimic was transfected into h1299 cells with or without target protector. At 48 h after transfection, <i>FZD2</i> mRNA levels were measured by real-time RT-PCR. β-Actin was used as an internal reference gene. Experiments were performed in triplicate, and relative <i>FZD2</i> mRNA (zeo = 100%) were averaged. Data are presented as the means ± SDs.</p

Effects of <i>miR-338-3p</i> and <i>miR-203</i> on the expression of <i>FZD</i> mRNA.

<p>h1299 cells were transfected with miRNA mimics or miRNA hairpin inhibitors, and total RNA was isolated after 48 h. mRNA levels were then analyzed by real-time RT-PCR. β-Actin was used as an internal reference gene. Experiments were performed in triplicate, and relative miRNA levels (zeo/control inhibitor = 1) were averaged. Data are presented as the means ± SDs.</p

<i>miR-203</i> targeted the <i>FZD2</i> gene.

<p>The FZD2 3′-UTR sequence and complementary <i>miR-203</i>-binding sequences are shown.</p

Effects of <i>miR-338-3p</i> on the expression of Frizzled (FZD) proteins.

<p>h1299 cells (h1299/zeo, h1299/CD82) were transfected with <i>miR-338-3p</i> mimic, <i>miR-338-3p</i> inhibitor, or miR controls. At 48 h after transfection, total protein was extracted and analyzed by immunoblot analysis with anti-FZD antibodies. The same blots were stripped and reprobed forβ-actin as a loading control. Experiments were repeated three times, and the most representative data are shown.</p