Abstract
The Microphthalmia associated transcription factor (Mitf) is an important regulator in melanocyte development and has been shown to be involved in melanoma progression. The current model for the role of Mitf in melanoma assumes that the total activity of the protein is tightly regulated in order to secure cell proliferation. Previous research has shown that regulation of Mitf is complex and involves regulation of expression, splicing, protein stability and post-translational modifications. Here we show that microRNAs (miRNAs) are also involved in regulating Mitf in melanoma cells. Sequence analysis revealed conserved binding sites for several miRNAs in the Mitf 3′UTR sequence. Furthermore, miR-148 was shown to affect Mitf mRNA expression in melanoma cells through a conserved binding site in the 3′UTR sequence of mouse and human Mitf. In addition we confirm the previously reported effects of miR-137 on Mitf. Other miRNAs, miR-27a, miR-32 and miR-124 which all have conserved binding sites in the Mitf 3′UTR sequence did not have effects on Mitf. Our data show that miR-148 and miR-137 present an additional level of regulating Mitf expression in melanocytes and melanoma cells. Loss of this regulation, either by mutations or by shortening of the 3′UTR sequence, is therefore a likely factor in melanoma formation and/or progression.
Highlights
The MITF (Microphthalmia associated transcription factor) protein is a master regulator of melanocyte development and is important in several other cell types including the retinal pigment epithelium (RPE) of the eye, osteoclasts and mast cells [1]
We tested the effects of microRNAs which have conserved binding sites in the 39UTR region of Microphthalmia associated transcription factor (Mitf), including miR-27a, miR-25/ 32/92/363/367 (1491–1497), miR-101/144 (3023–3029), miR124/506 (1639–1646) and miR-148/152 (1674–1680 and 2931– 2937) (Fig. 1A and 1B). miR-124/506 has a less conserved binding site at 548–554, and was tested in our study
The potential binding sites for miR-137 were tested as the mouse and human Mitf 39UTR sequences both contain two well conserved miR-137 binding sites located in close proximity to each other, 137C (2842–2848) and 137D (3061–3067)
Summary
The MITF (Microphthalmia associated transcription factor) protein is a master regulator of melanocyte development and is important in several other cell types including the retinal pigment epithelium (RPE) of the eye, osteoclasts and mast cells [1]. The Mitf gene encodes a bHLH-Zip transcription factor of the Myc family [3,4] which regulates melanocyte differentiation by activating other known melanocyte determinants, including Tyrosinase (Tyr), Tyrosinase-related protein-1 (Tyrp-1) and DCT/Tyrp-2 [5,6]. Mitf is regulated at multiple levels, including transcription, alternative splicing, post-translational modifications and protein stability. MITF is modified post-transcriptionally and has been shown to be phosphorylated at multiple sites leading to either increase in transcriptional activation or decrease in protein stability [14,15,16,17]. Ubiquitination marks MITF for degradaton [18] and sumoylation has been shown to affect DNA binding specificity [19,20]
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