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.
Although p53 is inactivated by point mutations in many tumors, melanomas infrequently harbor mutations in the p53 gene.,Here we investigate the biological role of microRNA-18b (miR-18b) in melanoma by targeting the MDM2-p53 pathway.,Expression of miR-18b was examined in nevi (n = 48) and melanoma (n = 92) samples and in melanoma cell lines and normal melanocytes.,Immunoblotting was performed to determine the expression of various proteins regulated by miR-18b.,The effects of miR-18b overexpression in melanoma cell lines were investigated using assays of colony formation, cell viability, migration, invasion, and cell cycle and in a xenograft model (n = 10 mice per group).,Chromatin immunoprecipitation and methylation assays were performed to determine the mechanism of microRNA silencing.,Expression of miR-18b was substantially reduced in melanoma specimens and cell lines by virtue of hypermethylation and was reinduced (by 1.5- to 5.3-fold) in melanoma cell lines after 5-AZA-deoxycytidine treatment.,MDM2 was identified as a target of miR-18b action, and overexpression of miR-18b in melanoma cells was accompanied by 75% reduced MDM2 expression and 2.5-fold upregulation of p53, resulting in 70% suppression of melanoma cell colony formation.,The effects of miR-18b overexpression on the p53 pathway and on melanoma cell growth were reversed by MDM2 overexpression.,Stable overexpression of miR-18b produced potent tumor suppressor activity, as evidenced by suppressed melanoma cell viability, induction of apoptosis, and reduced tumor growth in vivo. miR-18b overexpression suppressed melanoma cell migration and invasiveness and reversed epithelial-to-mesenchymal transition.,Our results demonstrate a novel role for miR-18b as a tumor suppressor in melanoma, identify the MDM2-p53 pathway as a target of miR-18b action, and suggest miR-18b overexpression as a novel strategy to reactivate the p53 pathway in human tumors.