Metastatic melanoma is the most aggressive form of this cancer.,It is important to understand factors that increase or decrease metastatic activity in order to more effectively research and implement treatments for melanoma.,Increased cell invasion through the extracellular matrix is required for metastasis and is enhanced by matrix metalloproteinases (MMPs).,Tissue inhibitor of metalloproteinases 3 (TIMP3) inhibits MMP activity.,It was previously shown by our group that miR-21, a potential regulator of TIMP3, is over-expressed in cutaneous melanoma.,It was therefore hypothesized that increased levels of miR-21 expression would lead to decreased expression of TIMP3 and thereby enhance the invasiveness of melanoma cells. miR-21 over-expression in the melanoma cell lines WM1552c, WM793b, A375 and MEL 39 was accomplished via transfection with pre-miR-21.,Immunoblot analysis of miR-21-overexpressing cell lines revealed reduced expression of TIMP3 as compared to controls.,This in turn led to a significant increase in the invasiveness of the radial growth phase cell line WM1552c and the vertical growth phase cell line WM793b (p < 0.05), but not in the metastatic cell lines A375 or MEL 39.,The proliferation and migration of miR-21 over-expressing cell lines was not affected.,Reduced expression of TIMP3 was achieved by siRNA knockdown and significantly enhanced invasion of melanoma cell lines, mimicking the effects of miR-21 over-expression.,Treatment of tumor cells with a linked nucleic acid antagomir to miR-21 inhibited tumor growth and increased tumor expression of TIMP3 in vivo in 01B74 Athymic NCr-nu/nu mice.,Intra-tumoral injections of anti-miR-21 produced similar effects.,This data shows that increased expression of miR-21 enhanced the invasive potential of melanoma cell lines through TIMP3 inhibition.,Therefore, inhibition of miR-21 in melanoma may reduce melanoma invasiveness.
Melanoma is an aggressive cancer that metastasizes rapidly, and is refractory to conventional chemotherapies.,Identifying miRNAs that are responsible for this pathogenesis is therefore a promising means of developing new therapies.,We identified miR-26a through microarray and qRT-PCR experiments as an miRNA that is strongly down-regulated in melanoma cell lines as compared to primary melanocytes.,Treatment of cell lines with miR-26a mimic caused significant and rapid cell death compared to a negative control in most melanoma cell lines tested.,In surveying targets of miR-26a, we found that protein levels of SMAD1 and BAG-4/SODD were strongly decreased in sensitive cells treated with miR-26a mimic compared to the control.,The luciferase reporter assays further demonstrated that miR-26a can repress gene expression through the binding site in the 3′UTR of SODD.,Knockdown of these proteins with siRNA showed that SODD plays an important role in protecting melanoma cells from apoptosis in most cell lines sensitive to miR-26a, while SMAD1 may play a minor role.,Furthermore, transfecting cells with a miR-26a inhibitor increased SODD expression.,Our findings indicate that miR-26a replacement is a potential therapeutic strategy for metastatic melanoma, and that SODD in particular is a potentially useful therapeutic target.