388 Exp. Mol. Med. Vol. 44(6), 387-393, 2012 Figure 1. Expression levels of ga- lectin-3 and MMP-1 in several mouse cell lines. (A) mRNA and (B) protein expression levels of ga- lectin-3 and MMP-1 in B16F10, 4T-1,TC-1 and NIH3T3 cell lines, determined by RT-PCR and western blotting. β-actin was used as a load- ing control. (C) mRNA and (D) pro- tein expression levels of galectin-3 and MMP-1 in B16F1 and B16F10 cell lines were determined by RT-PCR and western blotting. β-ac- tin was used as normalization control. studies showed that MMP-1 knockdown cells reduce melanoma invasion into matrigel, whereas its activation promoted melanoma cell invasion into three dimensional type I collagen (Durko et al., 1997; Benbow et al., 1999). Clinical studies have also provided evidence that MMP-1 is positively correlated with malignancy of melanoma cells and poor prognosis of patients (Nikkola et al., 2002). MMP-1 expression is controlled by mitogen activated protein kinase (MAPK) pathway(s) and the upstream transcription factor, activation of activator protein-1 (AP-1) (Tower et al., 2003; Jung et al., 2008). Although increasing attention has been paid to the roles of galectin-3 and MMP-1 in cancer metastasis, and a recent study showed a significant role of galectin-3 in MMP-1 expression in gastric cancer cells (Cheong et al., 2010; Cho et al., 2011; Kim et al., 2011), it is unclear whether similar regulation occurs in melanoma. In this study, we demonstrated that galectin-3 regulated MMP-1 expression and melanoma cell metastasis using B16F10 cell line, as a highly expression of both galectin-3 and MMP-1 in a mouse melanoma cell line. The B16F10 cell line is well established in in vivo mouse lung metastasis systems (Nguyen et al., 2007). We demonstrated that knockdown of galectin-3 reduced MMP-1 expression and melanoma metastasis both in vitro and in vivo, in part by modulating AP-1 activity. Results Expression levels of galectin-3 and MMP-1 in mouse cancer cell lines The mRNA and protein expression of galectin-3 and MMP-1 were assessed in the following 4 cell types: highly metastatic melanoma cancer cell (B16F10), highly metastatic breast cancer cell (4T-1), HPV positive and invasive mouse cervical cancer cells (TC-1), and mouse fibroblast cells (NIH3T3). As shown in Figures 1A and 1B, galectin-3 was highly expressed in all four cell lines, whereas the expression of MMP-1 was obviously higher in the B16F10 cell line than in others. Compared to the non-metastatic cell line B16F1, B16F10 cells express a higher level of both galectin-3 and MMP-1, in both mRNA and protein levels, respectively (Figures 1C and 1D). Galectin-3 knockdown inhibited B16F10 melanoma cell migration and invasion To examine the effects of galectin-3 knockdown on B16F10 cell migration and invasion, cells were transfected with various concentrations of galectin-3 siRNA, and galectin-3 expression was assessed using real time PCR and western blotting respectively. As shown in Figure 2A, all three concentrations of galectin-3 siRNA significantly reduced protein expression, with no significant differences in potency. Thus, 20 nM of galectin-3 was chosen for the following experiments. After transfection of B16F10 cells with 20 nM galectin-3 siRNA for 24 h, the living cells were collected and subjected to wound healing, invasion and migration assays. As shown in Figure 2B, cells transfected with galectin-3 siRNA migrated significantly slower than those without transfection or transfected with a scRNA in wound healing assays. Representative images are shown in Figure 2C. In addition, our trans-well migration and invasion assays also demonstrated approximately 50% and 65% decrease in cell migration and invasion, respectively, in the galectin-3 siRNA transfected cells compared to the control cells (Figures 2D and 2E).