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MicroRNA miR-196a is a central regulator of HOX-B7 and BMP4 expression in malignant melanoma

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Abstract

Since bone morphogenetic proteins (BMPs) play an important role in melanoma progression, we aimed to determine the molecular mechanisms leading to overexpression of BMP4 in melanoma cells compared to normal melanocytes. With our experimental approach we revealed that loss of expression of a microRNA represents the starting point for a signaling cascade finally resulting in overexpression of BMP4 in melanoma cells. In detail, strongly reduced expression of the microRNA miR-196a in melanoma cells compared to healthy melanocytes leads to enhanced HOX-B7 mRNA and protein levels, which subsequently raise Ets-1 activity by inducing basic fibroblast growth factor (bFGF). Ets-1 finally accounts for induction of BMP4 expression. We were furthermore able to demonstrate that bFGF-mediated induction of migration is achieved via activation of BMP4, thus determining BMP4 as major modulator of migration in melanoma. In summary, our study provides insights into the early steps of melanoma progression and might thereby harbor therapeutic relevance.

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Acknowledgments

This work was supported by the Deutsche Krebshilfe e.V. (grant number 108777) and the Deutsche Forschungsgemeinschaft (grant number BO1573/16-1). We thank Prof. Soncin (Lille, France) for providing the Ets-1 construct, Dr. Madry (Homburg, Germany) for providing the bFGF expression plasmid; Dr. Moser (Martinsried, Germany) for providing the BMP4 promoter reporter constructs; and Sibylla Lodermeyer for excellent technical assistance.

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  1. Institute of Pathology, University of Regensburg Medical School, Franz-Josef-Strauss-Allee 11, 93053, Regensburg, Germany

    Simone Braig, Daniel W. Mueller, Tanja Rothhammer & Anja-Katrin Bosserhoff

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  1. Simone Braig
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  2. Daniel W. Mueller
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  3. Tanja Rothhammer
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  4. Anja-Katrin Bosserhoff
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Correspondence to Anja-Katrin Bosserhoff.

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S. Braig and D. W. Mueller contributed equally to this work.

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18_2010_394_MOESM1_ESM.jpeg

Fig. S1 Confirmation of bFGF overexpression in Mel Im melanoma cells transduced with a bFGF expressing adenovirus by qRT-PCR. Bars show the means ± SD of three independent experiments, measurements were performed in duplicates (***, p< 0.001; **, p< 0.01; *, p< 0.5) (JPEG 229 kb)

18_2010_394_MOESM2_ESM.jpeg

Fig. S2 Compilation of photographs exemplary for filter areas counted in the Boyden chamber assays described in Fig. 1C (JPEG 2028 kb)

18_2010_394_MOESM3_ESM.tif

Fig. S3 Enhanced migratory potential of NHEMs in Boyden chamber assays after treatment with recombinant BMP4 protein (100ng/ml) for 24h. Untreated NHEMs served as a control. Bars show the means ± SD of one out of three independent experiments, measurements were performed in triplicates (***, p< 0.001; **, p< 0.01; *, p< 0.5) (TIFF 10503 kb)

18_2010_394_MOESM4_ESM.jpeg

Fig. S4 (A) bFGF and (B) BMP4 protein expression in two cell lines derived from primary melanoma lesions and two cell lines derived from metastatic melanomas, respectively. Absolute protein levels were measured by ELISA. Protein levels of bFGF and BMP4 in standard DMEM medium were determined and subsequently subtracted from total values. After subtraction of bFGF and BMP4 levels measured in the melanocyte growth medium used, NHEMs showed no endogenous expression of bFGF and BMP4, respectively. One representative experiment is shown out of three replicates (JPEG 1159 kb)

18_2010_394_MOESM5_ESM.jpeg

Fig. S5 HOX-B7 mRNA expression is reduced in the melanoma cell line Mel Im after transfection of either siHOX-B7#1 or siHOX-B7#4 compared to control transfected cells as shown by quantitative RT-PCR. Bars show the means ± SD of three independent experiments, measurements were performed in duplicates (***, p< 0.001; **, p< 0.01; *, p< 0.5) (JPEG 629 kb)

18_2010_394_MOESM6_ESM.jpeg

Fig. S6 Absolute bFGF and BMP4 protein levels of Mel Im cells transfected with siRNA against HOX-B7 for 48h. One representative out of three independent experiments is shown (JPEG 615 kb)

18_2010_394_MOESM7_ESM.jpeg

Fig. S7 The miR-196a re-expressing cell clones exhibited decreased Id1 mRNA expression compared to pcDNA3 empty vector control cells as shown by quantitative RT-PCR. Bars show the means ± SD of three independent experiments, measurements were performed in duplicates (***, p< 0.001; **, p< 0.01; *, p< 0.5) (JPEG 632 kb)

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Braig, S., Mueller, D.W., Rothhammer, T. et al. MicroRNA miR-196a is a central regulator of HOX-B7 and BMP4 expression in malignant melanoma. Cell. Mol. Life Sci. 67, 3535–3548 (2010). https://doi.org/10.1007/s00018-010-0394-7

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