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Venza M.,Azienda Policlinico Universitario rtino | Visalli M.,Azienda Policlinico Universitario rtino | Catalano T.,Azienda Policlinico Universitario rtino | Biondo C.,Azienda Policlinico Universitario rtino | And 4 more authors.
Oncology Reports

E-cadherin, a calcium-dependent cell-cell adhesion molecule, has an important role in epithelial cell function, maintenance of tissue architecture and cancer suppression. Loss of E-cadherin promotes tumor metastatic dissemination and predicts poor prognosis. The present study investigated the clinicopathological significance of E-cadherin expression in cutaneous, mucosal and uveal melanoma related to epigenetic mechanisms that may contribute to E-cadherin silencing. E-cadherin expression was reduced in 55/130 cutaneous (42.3%), 49/82 mucosal (59.7%) and 36/64 uveal (56.2%) melanoma samples as compared to normal skin controls and was inversely associated with promoter methylation. Of the 10 different CpG sites studied (nt 863, 865, 873, 879, 887, 892, 901, 918, 920 and 940), two sites (nt 892 and 940) were 90-100% methylated in all the melanoma specimens examined and the other ones were partially methylated (range, 53-86%). In contrast, the methylation rate of the E-cadherin gene was low in normal tissues (range, 5-24%). In all the three types of melanoma studied, a significant correlation was found between reduced levels of E-cadherin and reduced survival, high mitotic index and metastasis, accounting for the predilection of lymph nodal localization. In cutaneous and mucosal melanoma, low E-cadherin expression was positively correlated also with head/neck localization and ulceration. A high frequency of reduced E-cadherin levels occurred in choroid melanomas. In vitro experiments showed that E-cadherin transcription was restored following 5-aza-2'-deoxycytidine (5-aza-dC) treatment or DNMT1 silencing and was negatively correlated with the invasive potential of melanoma cells. The significant relationship between E-cadherin silencing and several poor prognostic factors indicates that this adhesion molecule may play an important role in melanomagenesis. Therefore, the inverse association of E-cadherin expression with promoter methylation raises the intriguing possibility that reactivation of E-cadherin expression through promoter demethylation may represent a potential therapeutic strategy for the treatment of melanoma. Source

Venza I.,Azienda Policlinico Universitario rtino | Visalli M.,Azienda Policlinico Universitario rtino | Beninati C.,Messina University | Benfatto S.,Azienda Policlinicouniversitario rtino | And 2 more authors.
BMC Medical Genomics

Background: IL-10 is an immunoregulatory cytokine that increases during malignant diseases. The purpose of this study was to: i) determine the mRNA amounts of IL-10, IL-10Raα, and IL-10Rβ in cutaneous and uveal melanoma cells and specimens; ii) evaluate their post-transcriptional regulation by miRNAs; iii) ascertain whether miRNA dysregulation may affect IL-10-induced proliferation. Methods: Genome-wide miRNA expression profiling was performed using a human microarray platform. The reference gene mRNA was measured through qPCR. miRNAs/mRNAs interactions were predicted by TargetScan, microRNA, and PITA. Transfections of specific miRNA mimics/inhibitors were carried out. Cell proliferation was assessed by MTT assay in the presence of IL-10 after transfection with miRNA mimics/inhibitors. Results: There were no differences in IL-10 mRNA levels between any of the 3 melanoma cell lines tested and normal melanocytes. However, lower IL-10Raα expression was found in G361 and OCM-1 cells, and higher levels of IL-10Rβ were observed in G361 cells compared with normal melanocytes. GR-M cells did not exhibit any modifications in IL-10Raα and IL-10Rβ expression. miR-15a, miR-185, miR-211, and miR-30d were upregulated in G361 and OCM-1 cells, remaining at similar levels in GR-M cells. miR-409-3p and miR-605were down-regulated exclusively in G361 cells. Prediction tools revealed that miR-15a, miR-185, and miR-211 targeted IL-10Raα whereas none of the miRNAs exclusively downregulated in G361 cells targeted IL-10Rβ. Luciferase reporter and western blot assays showed that IL-10Raα expression is directly regulated by miR-15a, miR-185, and miR-211, either alone or in combination. An inverse expression pattern between IL-10Raα, on one side, and miR-15a, miR-185, and miR-211 on the other one was also shown in melanoma samples. Ectopic expression of individual miR-15a, miR-185, and miR-211, and even more their co-expression, caused a marked decrease in the proliferation rate of all the cell lines. Likewise, inhibition of any specific miRNA promoted cell growth, an effect that further increased when inhibition concerned all three miRNA. Moreover, specific knockdown of IL-10Raα prevented the proliferative effect of miRNA inhibitors. Conclusions: Our results support a key role of IL-10Raα in the development and progression of melanoma and suggest that the IL-10/IL-10 receptor system may become a new therapeutic target for melanoma treatment. © 2015 Venza et al. Source

Venza M.,Messina University | Dell'Aversana C.,CNR Institute of Neuroscience | Dell'Aversana C.,Azienda Policlinico Universitario rtino | Visalli M.,Azienda Policlinico Universitario rtino | And 4 more authors.

Aims and background.microRNA (miRNA)-mediated epigenetic regulation of tumor suppressor genes and oncogenes has been shown to play a central role in melanomagenesis. Here, we focused on the identification of miRNA signatures in the cutaneous melanoma cell line G361 and the uveal melanoma cell line OCM-1. Methods and study design.We carried out genome-wide miRNA expression profiling using a human miRNA microarray platform (Agilent Sanger miRBase, release 10.1) in both cell lines. Results. Our screening revealed significant alteration of miRNA expression profiles in melanoma cell lines compared with normal human epidermal melanocytes. We defined 208 differentially expressed miRNAs in OCM-1 and 112 in G361. By comparison analysis between the resulting miRNA expression profiles, we identified 96 miRNAs that were modified in both cell models. Among these commonly modified miRNAs, 65 were downregulated, 28 upregulated, and 3 exhibited a different expression trend. Conclusions. Although preliminary, our analysis identified new melanoma-associated miRNAs providing novel miRNA candidates for the development of anticancer target therapy. Source

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