MicroRNA-365 (miR-365) is involved in the development of a variety of cancers.,However, it remains largely unknown if and how miRNAs-365 plays a role in melanoma development.,In this study, we overexpressed miR-365 in melanoma cell lines A375 and A2058, via transfection of miR-365 mimics oligos.,We then investigated alterations in a series of cancer-related phenotypes, including cell viability, cell cycle, apoptosis, colony formation, and migration and invasion capacities.,We also validated cyclin D1 (CCND1) and BCL2 apoptosis regulator (BCL2) as direct target genes of miR-365 by luciferase reporter assay and investigated their roles in miR-365 caused phenotypic changes.,To get a more general view of miR-365’s biological functions, candidate target genes of miR-365 were retrieved via searching online databases, which were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses for potential biological functions.,We then analyzed The Cancer Genome Atlas (TCGA) Skin Cutaneous Melanoma (SKCM) dataset for correlation between miR-365 level and clinicopathological features of patients, and for survival of patients with high and low miR-365 levels.,We found that miR-365 was downregulated in melanoma cells.,Overexpression of miR-365 remarkably suppressed cell proliferation, induced cell cycle arrest and apoptosis, and compromised the migration and invasion capacities in A375 and A2058 cell lines.,We also found that the phenotypic alterations by miR-365 were partially due to downregulation of CCND1 and BCL2 oncogenes.,The bioinformatics analysis revealed that predicted targets of miR-365 were widely involved in transcriptional regulation and cancer-related signaling pathways.,However, analysis of SKCM dataset failed to find differences in miR-365 level among melanoma patients at different clinicopathologic stages.,The Kaplan-Meier analysis also failed to discover significant differences in overall survival and disease-free survival between patients with high and low miR-365 levels.,Our findings suggested that miR-365 might be an important novel regulator for melanoma formation and development, however, the in vivo roles in melanoma developments need further investigation.
Micro RNAs (miRs) have emerged as key regulators during oncogenesis.,They have been found to regulate cell proliferation, differentiation, and apoptosis.,Mir-125b has been identified as an oncomir in various forms of tumours, but we have previously proposed that miR-125b is a suppressor of lymph node metastasis in cutaneous malignant melanoma.,Our goal was therefore to further examine this theory.,We used in-situ-hybridization to visualise miR-125b expression in primary tumours and in lymph node metastasis.,Then using a miRVector plasmid containing a miR-125b-1 insert we transfected melanoma cell line Mel-Juso and then investigated the effect of the presence of a stable overexpression of miR-125b on growth by western blotting, flow cytometry and β-galactosidase staining.,The tumourogenicity of the transfected cells was tested using a murine model and the tumours were further examined with in-situ-hybridization.,In primary human tumours and in lymph node metastases increased expression of miR-125b was found in single, large tumour cells with abundant cytoplasm.,A stable overexpression of miR-125b in human melanoma cell line Mel-Juso resulted in a G0/G1 cell cycle block and emergence of large cells expressing senescence markers: senescence-associated beta-galactosidase, p21, p27 and p53.,Mel-Juso cells overexpressing miR-125b were tumourigenic in mice, but the tumours exhibited higher level of cell senescence and decreased expression of proliferation markers, cyclin D1 and Ki67 than the control tumours.,Our results confirm the theory that miR-125b functions as a tumour supressor in cutaneous malignant melanoma by regulating cellular senescence, which is one of the central mechanisms protecting against the development and progression of malignant melanoma.