Highly expressed miR-375 is not an intracellular 2 oncogene in Merkel cell polyomavirus-associated 3 Merkel cell carcinoma 4

miR-375 is a highly abundant miRNA in Merkel cell carcinoma (MCC), in other cancers it 16 acts either as a tumor suppressor or oncogene. While free-circulating miR-375 serves as surrogate 17 marker for tumor burden in patients with advanced MCC, its function within MCC cells has not 18 been established. Nearly complete miR-375 knockdown in MCC cell lines was achieved using 19 antagomiRs via nucleofection. Neither cell viability, growth characteristics nor morphology were 20 altered by this knockdown. miR-375 target genes and related signaling pathways were determined 21 using ENCORI revealing Hippo signaling and EMT-related genes likely to be regulated. Thus, their 22 expression was analyzed by multiplexed qRT-PCR after miR-375 knockdown demonstrating only a 23 limited change in expression. In summary, highly effective miR-375 knockdown in classical MCC 24 cell lines did neither significantly change cell viability, morphology, nor oncogenic signaling 25 pathways. These observations render miR-375 an unlikely intracellular oncogene in MCC cells, thus 26 suggesting to address likely functions of miR-375 for intercellular communication of MCC. 27