G361 cutaneous melanoma cells (ECACC, European Collection of Cell Cultures, Salisbury, UK) were grown in McCoy’s 5a medium modified with 10 % FBS, 2 mM L-glutamine, and 1 % penicillin/streptomycin. The cutaneous melanoma cell line GR-M (ECACC, European Collection of Cell Cultures, Salisbury, UK) and the uveal melanoma cell line OCM-1 (provided by J. Mellon, Department of Ophthalmology, UT Southwestern Medical Center, Dallas, TX) were cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 1 % penicillin/streptomycin, and 10 % FBS.
To exclude that our observations were restricted to cell lines maintained in long-term culture, we also examined primary tumors. Formalin-fixed paraffin-embedded (FFPE) tissue sections of 52 cutaneous melanomas (31 females and 21 males, age ranging from 45 to 65 years), 41 uveal melanomas (15 females and 26 males, age ranging from 52 to 68 years), and 35 normal skin specimens, taken from patients of the University Hospital of Messina, were examined. The investigation adhered to the Declaration of Helsinki and was approved by the Ethics Committee of the University Hospital of Messina. An informed verbal consent was given by patients.
Total RNA extraction, reverse transcription and qPCR
Total RNA was extracted by TRIzol Reagent (Invitrogen). RNA samples were quantified with a Nanodrop 1000 spectrophotometer and their quality was evaluated using Agilent RNA 6000 Nano Assay. RNA samples were converted into cDNA using IMProm-II™ reverse transcriptase kit (Promega). Quantitative Real-Time PCR was performed by ABI Prism 7500 Real-Time PCR System (Applied Biosystems, Milan, Italy) with SYBR Green Mastermix (Applied Biosystems, UK). Primers were previously reported [12, 13]. The mRNA levels of IL-10, IL-10Rα, and IL-10Rβ were normalized to endogenous β-actin (Applied Biosystems).
miRNA microarray experiments were performed using a human miRNA microarray platform (Agilent Sanger miRBase, release 10.1) with 723 human and 76 human viral miRNAs represented. Agilent Feature Extraction Software was used for background subtraction. LOWESS and Quantile normalizations were performed.
MiR-15a, miR-185, and miR-211 mimics/inhibitors (Qiagen, Milan, Italy) were transfected either alone or in combination into melanoma cells using HiPerFect according to the manufacturer’s protocols (Qiagen). Cells were transfected twice with 100 pmol of oligonucleotide per well (0.5 × 106 cells) at 24 h intervals. Transfected cells were assayed 48 h after the second transfection.
Western blot analysis
Total cell extracts (50 μg) were resolved by SDS-PAGE and blotted onto nitrocellulose membranes with specific antibodies against IL-10 (sc-8438, Santa Cruz Biotechnology), IL-10Rα (sc-984, Santa Cruz Biotechnology), and IL-10Rβ (sc-514822, Santa Cruz Biotechnology).
Plasmids constructs and transient transfections
The 3′-UTR of IL-10Rα and a mutation sequence were amplified by PCR using the primers with a Bgl II restriction site on each 5′ or 3′ strand. The PCR products were inserted into the Bgl II sites of the pGL3-control vector (Promega, Madison, WI, USA) and identified by DNA sequencing. The wild-type plasmids were created containing the 3′-UTR of IL-10Rα with complementary sequence of miR-15α ( IL-10Rα 3′-UTR wild 1), miR-185 (IL-10Rα 3′-UTR wild 2), miR-211 ( IL-10Rα 3′-UTR wild 3) or covering all the three miRNA side sites (IL-10Rα 3′-UTR wild-full-length). Four mutant plasmids were generated with the mutation sequence without complementary sequence of miR-15a (pGL3- IGF-1 3′-UTR mut 1), miR-185 (pGL3- IGF-1 3′-UTR mut 2), and miR-211 (pGL3- IGF-1 3′-UTR mut 3) or all of them (IL-10Rα 3′-UTR mut-full-length). For the luciferase reporter assay, cells were seeded on 24-well plates and co-transfected using Lipofectamine 2000 (Invitrogen) with 100 ng per well of the resulting luciferase UTR-report vector, 2 ng per well of pRLCMV vector (internal control, Promega), and 20 ng per well of miR-15a, miR-185, and miR-211 mimics or inhibitors following the manufacturer’s instructions (Qiagen, Milan, Italy). After 24 h, the cells were lysed, and the relative luciferase activity was assessed with the Dual-Luciferase Assay Reporter System (Promega). For transient knockdown of IL-10Rα, cells were transfected with specific small interfering RNA (siRNA) targeting IL-10Rα or non-targeting control siRNA (Santa Cruz Biotechnology, Milan, Italy) 24 h after plating using Lipofectamine 2000 (Invitrogen) with the siRNA at a final concentration of 100 nM.
Cell proliferation assay
Cells were treated with recombinant human IL-10 (R & D Systems) at various doses (50, 100, or 500 U/ml) and for different times (at 6-hr intervals during a 72-hr culture period) accordingly to previously reported conditions . Proliferation was measured using the MTT Assay Kit (Cayman Chemical Company, Michigan, USA).
Densitometry and statistical analysis
The one-way analysis of variance (ANOVA) test, followed by a pair-wise multiple comparison test (Bonferroni t test), was performed to identify the differences among the groups. The relative intensities of protein bands were analyzed by Image J software (Bethesda, MD, USA). Statistical significance was assigned when the p value was <0.05.