Figure 2. Effect of galectin-3 silencing on the migration and invasion of B16F10 melanoma cells. (A) mRNA and protein levels of galectin-3 after transfection of B16F10 melanoma cells with different concentrations (10, 20, 30 nmol/L) of scrambled siRNA (scRNA, negative control) and ga- lectin-3 siRNA. Total RNA and protein were obtained after transfection for 48 h, and cells were harvested and analyzed by real time-PCR and west- ern blotting, respectively. β-actin was used as a loading control. (B-C) A wound healing assay of B16F10 cells was performed with scRNA or ga- lectin-3 siRNA at 0, 12, 18 and 24h after scratching. (B) Quantification of cell migration as described in the Methods section and (C) the micro- graphs of B16F10 cells were obtained according to the width change of the area lacking cells (Bar = 100 μm). (D-E) Cell migration and invasion activities were measured respectively by a Transwell assay after trans- fection of melanoma cells with scRNA or galectin-3 siRNA for 24 h. All experiments were performed individually in triplicate (P ＜ 0.001 vs. con- trol group). Galectin-3 modulated MMP-1 expression through interacting with AP-1 Given the expressions of both galectin-3 and MMP-1 in the mouse B16F10 cell line, we examined Galectin-3 increases MMP-1 expression 389 their possible interactions and the effect of MMP-1 on cell motility. MMP-1 knockdown had no effect on the expression of galectin-3，but galectin-3 knock- down resulted a marked reduction of MMP-1 expression, indicating that galectin-3 is an upstream regulator of MMP-1 (Figure 3A). A cell proliferation assay demonstrated that transfection of gal-3 siRNA or MMP-1 siRNA slightly decreased cell proliferation compared to the scrambled siRNA transfected cells (Figure 3B). In contrast, MMP-1 knockdown dramatically decreased cell motility (Figures 3C and 3D). To further explore how galectin-3 increases MMP-1 expression in melanoma cells, we focused on the transcription factor, activator protein-1 (AP-1), which is a dimer composed of Jun and Fos-like region antigen (Fra-1) (Angel and Karin, 1991). The direct interaction between galectin-3 and members of AP-1, c-Jun and Fra-1 was demonstrated by immunoprecipitation (Figure 3E). Next, we assessed transcription activity of AP-1 using a luciferase assay. Compared to the control cells, transfection of the cells with galectin-3 shRNA significantly reduced AP-1 transcription activity (Figure 3F). Finally, we examined DNA binding activity of AP-1 with or without galectin-3. Figure 3G shows the binding site of the AP-1 complex on the MMP-1 promoter region. Our ChIP assay demon- strated that c-jun and fra-1 bound to the promoter regions of MMP-1 in the presence, but not in the absence, of galectin-3 (Figure 3H). Galectin-3 knockdown decreased B16F10 metastasis in lung metastasis mouse models We constructed lentiviral vectors containing lacZ (negative control) or galectin-3 shRNA, and infected B16F10 cells with the lentivirus. As shown in Figure 4A, transfection of the B16F10 cells with galectin-3 siRNA resulted in approximately 53% reduction of the mRNA level of the target protein. As expected, transfection of B16F10 cells with galectin-3 markedly reduced the expression of MMP-1 (Figure 4B). To examine the effect of galectin-3 knockdown on melanoma metastasis in vivo, we applied the lentiviral infected cells to a mouse pulmonary metastasis model. Metastasis was assessed in C57BL/6 mice four weeks after tail vein injection of B16F10 parent cells, B16F10 cells infected with lentiviral lacZ, or gal-3shRNA. As shown in Figures 4C and 4D, the total numbers of lung metastatic nodule were reduced by approximately 50% in the animals injected with the B16F10 cells containing lentiviral gal-3 shRNA compared to the animals injected with B16F10 parent cells or lacZ expressing B16F10 cells.