Circulating cell-free(cf) microRNAs (miRNAs) have been reported to exist in plasma.,MicroRNA-210(miR-210) is known to play important roles in the tumor hypoxic state.,We hypothesized that the expression levels of cf-miR-210 in plasma would predict early clinical recurrence in melanoma patients.,A direct miRNA assay on plasma (RT-qPCR-DP) was developed to improve cf-miRNA assay logistics, eliminate RNA extraction, and reduce specimen amount required.,RNA was extracted from formalin-fixed paraffin-embedded (FFPE) melanoma tissues (n = 108) and assessed by RT-qPCR.,Plasma (10 μl; n = 264) was procured from AJCC Stage III/IV patients in phase III clinical trials.,A RT-qPCR-DP was performed to detect cf-miR-210.,MiR-210 was significantly higher in metastatic tumors compared to primary tumors.,Cf-miR-210 was significantly higher in melanoma patients versus healthy donor controls.,In serial bloods within individual patients, cf-miR-210 < 3 months prior to disease recurrence significantly increased compared to baseline levels (p = 0.012).,ROC curve analysis demonstrated that patients with elevated cf-miR-210 were more likely to have disease recurrence.,Moreover, cf-miR-210 increase significantly correlated with poorer prognosis (p < 0.001).,Lactate dehydrogenase (LDH) level was also assessed within patients, and the AIC values for proportional hazards regression models of cf-miR-210(120.01) and LDH (122.91) demonstrated that cf-miR-210 is a better recurrence indicator.,We concluded enhanced cf-miR-210 provides identification of early systemic melanoma recurrence.
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.