A Comprehensive Analysis of MicroRNA Expression During Human Keratinocyte Differentiation In Vitro and In Vivo Janosch Hildebrand, Martin Rütze, Nicole.

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A Comprehensive Analysis of MicroRNA Expression During Human Keratinocyte Differentiation In Vitro and In Vivo Janosch Hildebrand, Martin Rütze, Nicole Walz, Stefan Gallinat, Horst Wenck, Wolfgang Deppert, Adam Grundhoff, Anja Knott Journal of Investigative Dermatology Volume 131, Issue 1, Pages 20-29 (January 2011) DOI: 10.1038/jid.2010.268 Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 MicroRNAs (miRNAs) commonly up- or downregulated during keratinocyte (KC) differentiation in vitro or in vivo. (a, c) Venn diagrams of up- (a) and downregulated (c) miRNAs in neonatal normal human epidermal KCs (NHEKs) at 3 days (3d) and 7 days (7d) after calcium-induced differentiation, and in terminally differentiated skin KCs (total fractions). The total number of miRNAs subject to significant expression changes (see Tables 1 and 2 for details) in each entity is shown in square brackets. (b, d) Expression of commonly up- (b) or downregulated (d) miRNAs in calcium-treated relative to undifferentiated NHEK cells (set to 1), or in terminally differentiated (total fraction) KCs relative to epidermal stem cell fractions from skin. Expression was measured using TaqMan low-density arrays and data were normalized to U6 small nuclear RNA levels. Error bars represent standard deviations between six independent calcium-induced differentiation experiments or between skin biopsies from five different donors. Journal of Investigative Dermatology 2011 131, 20-29DOI: (10.1038/jid.2010.268) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Functional validation of target genes regulated by multiple microRNAs (miRNAs). (a–c) 3′-Untranslated region (UTR)–luciferase assays revealing repression of the endothelin A receptor (EDNRA) (a), eyes absent homolog 4 (EYA4) (b), and ganglioside-induced differentiation-associated protein 1 (GDAP1) 3′-UTRs (c) by miRs-27b, -224, -26a, -200a, or -203. The miRNAs were transfected at a concentration of 100nM each either individually (leftmost bars in each panel) or in combination (bars labeled “all”). A scrambled miRNA mimic (Scr) served as a negative control. Repression is indicated as the percentage of luciferase activity seen with the negative control (100nM). Error bars represent standard deviations between three independent experiments. *P<0.05 and **P<0.01 (t-test). (d, e) Immunofluorescence staining of human cryofixed skin sections. EDNRA (d) localizes to basal cells, and EYA4 (e) is predominantly detected in keratinocytes located in niches of papillae. Bar=100μm. Journal of Investigative Dermatology 2011 131, 20-29DOI: (10.1038/jid.2010.268) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 MicroRNA-203 (miR-203) and miR-23b are differentiation markers of human skin keratinocytes (KCs) in vivo. (a) Expression of miR-203 and -23b in calcium-treated neonatal normal human epidermal KCs (NHEKs) relative to undifferentiated NHEK cells, and in terminally differentiated (total fraction) KCs relative to epidermal stem cell fractions from human skin. Error bars represent standard deviations between six independent calcium-induced differentiation experiments or skin biopsies from five different donors, respectively. (b, c) Digoxigenin-labeled LNA-modified nucleotide probes were used to detect miR-203 (b) and miR-23b (c) transcripts in human skin by in situ hybridization. A scrambled probe (d) was used as a control. Skin sections were treated with anti-digoxigenin–alkaline phosphatase conjugates to detect miRNA expression (blue) and counterstained with nuclear fast red. Representative pictures are shown. Bar=50μm. 7d, 7 days. Journal of Investigative Dermatology 2011 131, 20-29DOI: (10.1038/jid.2010.268) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions