Signature of VDR miRNAs and Epigenetic Modulation of Vitamin D Signaling in Melanoma Cell Lines

Materials and Methods

Cell culture. The human melanoma cell lines MeWo, SK-Mel-28, SK-Mel-5 and IGR (Department of Dermatology, Saarland University Hospital) were cultivated in RPMI media supplemented with 10% foetal calf serum (FCS) and 1% L-glutamine. Primary normal human melanocytes (NHM) were isolated from human foreskin and cultivated in serum free melanocyte growth medium M2 (Ready-to-use, PromoCell®, Heidelberg, Germany), after the addition of Melanocyte Growth Medium M2 supplement mix (C-39420) (PromoCell®). 1,25(OH)₂D₃ and TSA (Sigma-Aldrich, Taufkirchen, Germany) were dissolved in ethanol as stock solutions (4 mM and 1 mg/ml, respectively) and stored in the dark at −20°C, 5-Aza (Sigma-Aldrich) was dissolved in PBS (10 mM). Media containing varying concentrations of 1,25(OH)₂D₃, TSA or 5-Aza and their combinations were added to cultured cells 24 h after seeding the cells (to ensure semi confluent density) in 10 cm tissue culture dishes (Greiner Bio-One GmbH, Frickenhausen, Germany).

Apoptosis detection. Detection of apoptosis by FACS is based on the observation that soon after initiating apoptosis, most cell types translocate the membrane phospholipid phosphatidylserine (PS) from the inner face of the plasma membrane to the cell surface. Once on the cell surface, PS can be easily detected by staining with a fluorescent conjugate of Annexin V, a protein which has a strong natural affinity for PS. FACS was performed using an Annexin V-PE Apoptosis Detection Kit (Biovision Research Products, Mountain View, USA) according to the manufacturer's protocol. Cells were resuspended in 500 μl of 1× annexin V-PE binding buffer followed by adding 3 μl of Annexin V-PE and incubated at room temperature for 5 min in the dark. Apoptosis was measured using FACscan flow cytometry. Apoptosis detection by FACS is based on the observation that soon after initiating apoptosis, most cell types translocate the phospholipid phosphatidylserine (PS) from the inner face of the plasma membrane to the cell surface. Once at the cell surface, PS can be easily detected by staining with a fluorescent conjugate of annexin V, a protein which has a strong natural affinity for PS.

mRNA and miRNA isolation. RNA isolation was performed using a RNeasy Kit (Qiagen) according to the manufacturer's manual. The miRs were isolated using a miRNeasy Kit (Qiagen). The expression of VDR mRNA, miR-27b, and miR-125b were analyzed in the melanoma cell lines using real time PCR (LightCycler, Roche Diagnostic, Mannheim, Germany), 50 cycles.

Real time-PCR. RT reaction mixture was used as the template for real-time PCR, containing 2 μl of 10× VDR primers or 10× hypoxanthine guanine phosphoribosyl transferase (HPRT) primer (house keeping gene) purchased from Qiagen, (Catalogue number QT01010170, QT00059066). A final concentration of 1 mM MgCl₂ in the miR-125b or miR-27b RT reaction mixture was used as the template for real-time PCR, containing 2 μl of 10× miR-125b primer or 10x miR-27b primer and 10× RNU6B (RNU6B is a widely used endogenous reference RNA in many miRNA quantification studies because of its small size 45 nt), purchased from Qiagen (miScript Primer Assay and miScript universal primer).

miR-125b antisense. The IGR melanoma cells were seeded (to ensure semi confluent density) in 6 well plate in 2 ml RPMI media supplemented with 10%, 1% L-glutamine and 1% penicillin/streptomycin. The medium was changed after 24 h and transfection of the cells with miScript miRNA-125b inhibitor, miScript inhibitor (negative control) or HiPerFect transfection reagent (mock control), (Qiagen) was carried out according to the manufacturer's manuals. The miRNAs were isolated after 24 h from the media control, mock control, negative control, and miR-125b antisense wells.

Western blotting. The nuclear proteins were extracted using a NEPER® Nuclear and Cytoplasmic Extraction Reagents Kit (ThermoFisher Scientific) and Complete Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's manuals. Briefly the cell membrane was disrupted and the cytoplasmic contents were released then after recovering the intact nuclei from the cytoplasmic extract by centrifugation, the nuclei were lysed to yield the nuclear extract. The protein concentration was determined using a Bradford (Bio-Rad, Protein Assay, Bio-Rad Laboratories GmbH, Muenchen) protein assay and finally 40 μg of nuclear protein from each sample were used. Based on the finding that VDR protein level was relatively high in MCF7 breast cancer cell lines (10), we used this cell line as VDR protein positive control. After electrophoresis in 12.5% SDS-polyacrylamide gels, separated proteins were electrophoretically transferred to a PVDF membrane. Non-specific sites were blocked by 5% non-fat dry milk in PBS for 1 h at room temperature. The membrane was incubated with the primary antibody (VDR polyclonal (C-20): sc-1008 or VDR monoclonal (D-6): sc-13133, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C or with the primary antibody nucleolin (11) as a loading control for 1 h at room temperature in PBS containing 1% non-fat dry milk, washed three times with PBS for 10 min and incubated 1 h with the respective secondary antibody in PBS containing 1% non-fat dry milk. After washing, bands were visualized by chemiluminescence. The intensity of VDR band was analyzed using Bio-Rad Quantity One® 1-D analysis software in relation to the loading control gene band (nucleolin) in three independent experiments.

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Figure 1.

Baseline expression level of VDR mRNA, miR-125b and miR-27b.