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

Background: Melanoma cells express the nuclear vitamin D receptor (VDR), indicating that malignant melanoma represents a promising target for treatment with 1,25-dihydroxyvitamin D3 (1,25(OH) 2 D 3 ) or its analogs. We previously showed that some melanoma cell lines are resistant to the antiproliferative effects of 1,25(OH) 2 D 3 and that 1,25(OH) 2 D 3 -sensitivity can, at least in part, be restored by co-treatment with the histone deacetylase inhibitor (HDACI) Trichostatin A (TSA) or with the DNA methyltransferase inhibitor (DNMTI), 5-azacytidine (5-Aza). This study aimed at gaining further insights into the molecular mechanisms that underlie the epigenetic modulation of 1,25(OH) 2 D 3 -sensitivity in melanoma cells. Materials and Methods: The expression of VDR mRNA, protein and two candidates of VDR microRNAs (miR-125b, miR-27b) were compared in 1,25(OH) 2 D 3 - responsive (MeWo, SK-Mel28) and -resistant (SK-Mel5, IGR) melanoma cell lines and in normal human melanocytes (NHM) using real time PCR and western blot analysis. Additionally, the effect of 1,25(OH) 2 D 3 , epigenetic modulating drugs (TSA, 5-Aza) and miR-125b antisense on the expression of VDR messenger RNA (mRNA)/protein, miR-125b and miR- 27b was investigated. Results: Treatment with 1,25(OH) 2 D 3 and/or epigenetic drugs (5-Aza, TSA) modulated the VDR mRNA expression in the 1,25(OH) 2 D 3 -responsive and - resistant melanoma cell lines and in the NHM. Treatment with 5-Aza, but not with TSA, reduced the expression of miR-125b in the 1,25(OH) 2 D 3 -responsive and -resistant melanoma cell lines and in the NHM. Treatment with 1,25(OH) 2 D 3 and/or epigenetic drugs (5-Aza, TSA) reduced the miR-27b expression in three out of four melanoma cell lines. Moreover, no difference was observed in VDR protein expression in the 1,25(OH) 2 D 3 -responsive as compared to the 1,25(OH) 2 D 3 - resistant melanoma cell lines. Transfection with miR-125b antisense did not affect the VDR mRNA/protein expression in the IGR cells. Conclusion: Responsiveness to 1,25(OH) 2 D 3