BackgroundmiRNAs are small noncoding RNAs that function as posttranscriptional regulators during development and disease. Aberrant expression of miRNAs has been associated with various types of malignant tumors. Decreased levels of miR-124 have been observed in human cancers. RACK1 is a scaffold protein that acts as an oncogene in various human cancers. The association between miR-124 and RACK1 in melanoma has not been characterized.Materials and methodsReal-time quantitative PCR was used to analyze RACK1 and miR-124 expression in melanoma tissue and cell lines. Dual-Luciferase reporter assay was performed to evaluate the effect of miR-124 inhibition on RACK1 expression. The effects of miR-124 on RACK1 in melanoma cell lines were evaluated using Western blot analysis and immunocytochemical staining. Wound-healing, transwell, and MTT assays, and annexin V-fluorescein isothiocyanate/propidium iodide followed by flow cytometry were used to evaluate the effects of miR-124 on RACK1-mediated proliferation, migration, invasion, and apoptosis of melanoma cells.ResultsThe expression of miR-124 in melanoma tissue was lower than that in normal skin tissue, and the expression of RACK1 was higher in melanoma tissue than that in normal skin tissue. Analysis using Dual-Luciferase reporter assay showed that RACK1 was a direct target of miR-124. Western blot and immunocytochemical staining showed that the expression of RACK1 was significantly inhibited by miR-124 in both A375 and A875 melanoma cells. Furthermore, the results of functional experiments showed that degradation of RACK1 by miR-124 inhibited proliferation, migration, and invasion of melanoma cells, and promoted melanoma cell apoptosis.ConclusionThe results suggested that miR-124 affected melanoma cells by directly targeting RACK1. miR-124 and RACK1 may be biomarkers for clinical diagnosis, and prognostic factors of human melanoma. Furthermore, miR-124 and RACK1 may be targets for the treatment of melanoma.

Abstract. It has been demonstrated that numerous types of metastatic cancer overexpress vacuolar-type H + (V)-ATPases. It may be possible to inhibit the growth and metastasis of human cancer cells by inhibiting V-ATPases. It was previously reported that diphyllin, a novel V-ATPase inhibitor, can inhibit the migration and invasion of SGC7901 human gastric cancer cells; however, the effects of cleistanthin A (CA), a diphyllin glycoside, on melanoma cells has not been demonstrated. The present study aimed to investigate the effect of CA as a V-ATPase inhibitor and its effects on the invasion and metastasis of A375 cells. The results of an MTT assay in the present study indicated that the growth inhibition of A375 cells by CA was induced in a dose-and time-dependent manner; however, A375 cell viability was not significantly affected by low concentrations (0.03, 0.1 and 0.3 µM) after 24 h. Similar results were obtained by viable cell counting with trypan blue. Therefore, these concentrations of CA were selected for the treatment of A375 cells in further experiments. It was demonstrated that CA inhibited the expression of V-ATPases in a dose-dependent manner and decreased the internal pH level of A375 cells. Alterations to the lysosomal pH were associated with the CA concentration. Furthermore, CA treatment induced a significant decrease in cell migration and invasion, as demonstrated with wound-healing and Transwell assays. Gelatin zymography and western blot analysis demonstrated that the expression levels of matrix metallopeptidase (MMP)-2 and -9 decreased following CA treatment. Therefore, CA can be characterized as a novel V-ATPase inhibitor for the treatment of melanoma that may inhibit invasion and metastasis by downregulating the expression of MMP-2 and -9.

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Melanoma is a highly malignant skin cancer with limited treatment options, the mechanism of the occurrence and development of melanoma is still unclear till now. Receptor for activated C kinase 1 (RACK1) is a scaffolding protein that mediates multiple signaling pathways; it interconnects distinct signaling pathways to control essential cellular processes. RACK1 was reported as an oncogene in human tumorigenesis, but little is known about its role in melanoma. This study aimed to investigate the expression of RACK1 in patients with melanoma and to reveal its possible functions in melanoma cells. The expression profiles of RACK1 detected in tumor tissues from melanoma patients showed that RACK1 was higher in tumor tissues, and its expression level was well associated with the clinical progression of melanoma (TNM stage, P=0.009). Furthermore, RNA interfering (RNAi) knockdown of RACK1 could efficiently suppress the proliferation, migration and invasion of A375 and A875 cells and promote their apoptosis. Taken together, these results suggest that RACK1 may be a poor prognostic factor in human melanoma, and it may be a new therapeutic target for melanoma treatment.