Hsa-microRNA-34a-5p inhibits virus-negative Merkel cell carcinoma cell proliferation and migration by regulating cell cycle- and EMT-related pathways and suppresses tumor spheroid formation

Background: The molecular mechanisms of Merkel cell polyomavirus (MCPyV)-negative MCC (VN-MCC) initiation remain poorly understood. Although hsa-miR-34a-5p dysregulation has been reported in MCC, its role in VN-MCC is unknown. Objective: We aimed to investigate the functional role of hsa-miR-34a-5p on the malignant phenotype of VN-MCC and elucidate the potential underlying mechanisms. Methods: Hsa-miR-34a-5p expression was investigated in MCC cell lines (n = 5) and tissues (n = 34), and in fibroblast/epithelial cell lines (n = 2). Functional experiments evaluated the effect of hsa-miR-34a-5p on VN-MCC MCC13 phenotype. Gene expression profiling, enrichment analyses and protein–protein interaction network of hsa-miR-34a-5p target genes (n = 84) were conducted in these cells to identify relevant targets/pathways. The impact of hsa-miR-34a-5p on VN-MCC spheroid volume/growth was investigated. Results: Hsa-miR-34a-5p was significantly downregulated in VN-MCC cells and tissues compared to MCPyV-positive counterparts, as well as to fibroblast/epithelial cells. Mechanistically, ectopic hsa-miR-34a-5p expression in MCC13 cells significantly inhibited proliferation, colony formation, and migration abilities, while promoted apoptosis. Hsa-miR-34a-5p silencing in epithelial HaCaT cells increased colony formation and partially enhanced migration. Ectopic hsa-miR-34a-5p expression in MCC13 cells negatively regulated key target genes and pathways involved in both G1/S transition of the cell cycle (CDK4, CDK6, CCNE2) and epithelial-to-mesenchymal transition (MET, NOTCH1, JAG1, along with Snail protein), leading to anti-proliferative and anti-migratory effects. Ectopic hsa-miR-34a-5p expression strongly inhibited MCC13 spheroid formation, whereas miRNA inhibition yielded the opposite effect in HaCaT spheroids from intermediate through later time points. Conclusion: We provide the first functional evidence of the pleiotropic tumor suppressor role of hsa-miR-34a-5p in VN-MCC.