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Abstract

Melanoma is a fast progressing tumor which tends to metastasize at small size and early time point. miRNA profiling has been performed for a variety of cancer types, but data differ substantially between studies. Therefore, it is of main interest to unravel miRNA networks which are involved in main aspects of melanoma progression, such as cell invasion. In order to obtain candidate miRNAs relevant for the control of invasion, a high-throughput invasion assay in a 96-well Boyden chamber format was performed for functional screening of a human miRNA mimics library consisting of 988 miRNAs (miRBase Version 13.0). The identifed miRNAs could be categorized into miRNAs increasing the invasive capacity of A375 melanoma cells (oncogenic miRNAs) or miRNAs decreasing the invasiveness, which were predominantly downregulated in melanoma and could function as tumor suppressors. miRNA expression analysis was performed to further validate these candidate miRNAs for their physiological role in different melanoma cell lines. miR-339-3p was defined as one promising candidate inhibiting invasion when transfected into different melanoma cell lines. miR-339-3p was expressed to significantly lower extent in melanoma cell lines compared to normal human epidermal melanocytes. Furthermore, A375 cells stably overexpressing miR-339-3p showed decreased lung colonization in NSG mice in comparison to cells expressing the empty vector control. The myeloid leukemia cell differentiation protein (MCL1) was identifed as a potential target of miR-339-3p by target prediction analysis and could be confirmed experimentally: miR-339-3p transfection resulted in downregulation of MCL1 (i) protein and (ii) mRNA levels; (iii) miR-339-3p antagomiR treatment increased melanoma cell invasion; (iv) the direct interaction of miR-339-3p and MCL1 3'UTR was shown in a 3'UTR binding assay; (v)MCL1 downregulation by siRNA inhibited melanoma cell invasion to comparable extent as mediated by miR-339-3p transfection. These findings indicate that miR-339-3p can act as a tumor suppressor in melanoma and the extent of its expression levels might contribute to the varying aggressiveness of different melanomas. This presented approach may help to unravel possible therapeutic checkpoints within the miRNA network of malignant melanoma cells to counteract tumor spread.