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.


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.


CONCLUSIONS
Responsiveness to 1,25(OH)(2)D(3) corresponds to the expression level of VDR mRNA which in turn might be regulated by VDR microRNAs or epigenetic modulating drugs.
Department of Dermatology, The Saarland University Hospital, Kirrbergerstr, 66421 Homburg, Germany. ssessa77@hotmail.com
Salma
S
Essa
Sandra
S
Reichrath
Ulrich
U
Mahlknecht
Mathias
M
Montenarh
Thomas
T
Vogt
Jörg
J
Reichrath
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001374:Azacitidine / Q000494:pharmacology; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D057890:Epigenomics; D006801:Humans; D008544:Melanocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008545:Melanoma / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D018167:Receptors, Calcitriol / Q000235:genetics* / Q000378:metabolism; D012878:Skin Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000494:pharmacology
D000964:Antimetabolites, Antineoplastic; C531888:MIRN125 microRNA, human; C531388:MIRN27 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; D018167:Receptors, Calcitriol; C118756:dihydroxy-vitamin D3; D014807:Vitamin D; D001374:Azacitidine