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.
Long non-coding RNAs (lncRNAs) have been shown to be implicated in the complex network of cancer including malignant melanoma and play important roles in tumorigenesis and progression.,However, their functions and downstream mechanisms are largely unknown.,This study aimed to investigate whether BRAF-activated non-coding RNA (BANCR), a novel and potential regulator of melanoma cell, participates in the proliferation of malignant melanoma and elucidate the underlying mechanism in this process.,We found that BANCR was abnormally overexpressed in human malignant melanoma cell lines and tissues, and increased with tumor stages by quantitative PCR.,BANCR knockdown induced by shRNA transfection significantly inhibited proliferation of tumor cells and inactivated MAPK pathway, especially by silencing the ERK1/2 and JNK component.,Moreover, combination treatment of BANCR knockdown and suppression ERK1/2 or JNK (induced by specific inhibitors U0126 or SP600125 respectively) produced synergistic inhibitory effects in vitro.,And the inhibitory effects induced by ERK1/2 or JNK could be rescued by BANCR overexpression.,By tumorigenicity assay in BALB/c nude mice, we further found that BANCR knockdown inhibited tumor growth in vivo.,In addition, patients with high expression of BANCR had a lower survival rate.,Taken together, we confirmed the abnormal upregulation of a novel lncRNA, BANCR, in human malignant melanoma.,BANCR was involved in melanoma cell proliferation both in vitro and in vivo.,The linkage between BANCR and MAPK pathway may provide a novel interpretation for the mechanism of proliferation regulation in malignant melanoma.