Abstract
miR-375 is a highly abundant miRNA in Merkel cell carcinoma (MCC). In other cancers, it acts as either a tumor suppressor or oncogene. While free-circulating miR-375 serves as a surrogate marker for tumor burden in patients with advanced MCC, its function within MCC cells has not been established. Nearly complete miR-375 knockdown in MCC cell lines was achieved using antagomiRs via nucleofection. The cell viability, growth characteristics, and morphology were not altered by this knockdown. miR-375 target genes and related signaling pathways were determined using Encyclopedia of RNA Interactomes (ENCORI) revealing Hippo signaling and epithelial to mesenchymal transition (EMT)-related genes likely to be regulated. Therefore, their expression was analyzed by multiplexed qRT-PCR after miR-375 knockdown, demonstrating only a limited change in expression. In summary, highly effective miR-375 knockdown in classical MCC cell lines did not significantly change the cell viability, morphology, or oncogenic signaling pathways. These observations render miR-375 an unlikely intracellular oncogene in MCC cells, thus suggesting that likely functions of miR-375 for the intercellular communication of MCC should be addressed.
Highlights
Merkel cell carcinoma (MCC) is an aggressive skin cancer
We introduced the respective antagomiRs with two different transfection conditions, which revealed that nuclear transfection was much more efficient and only this method succeeded in nearly complete knockdown up to five days post-transfection
We have demonstrated that even the highly efficient, almost complete knockdown of the highly abundant miR-375 in classical MCC cells lines, has no relevant impact on the cell viability, metabolic activity, morphology, or oncogenic signaling pathways targeted by miR-375
Summary
Merkel cell carcinoma (MCC) is an aggressive skin cancer. Risk factors for MCC include an advanced age, ultraviolet (UV) light exposure, and immune suppression [1]. About 80% of MCC tumors are associated with genomic integration of the Merkel cell polyomavirus (MCPyV) bearing truncating tumor-specific large T antigen mutations, while the others are characterized by a UV-induced tumor mutational burden [1]. Recent reports from the same group showed that miR-375, together with other miRNAs, inhibits autophagy, protecting MCC cells from autophagy-associated cell death [28]. To resolve these controversies, here, we scrutinize the function of miR-375 in MCC. We established a highly efficient method for miR-375 knockdown in classical MCC cell lines and analyzed the inflected effects, with an emphasis on intracellular signaling
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