Psoriasis Skin Inflammation-Induced microRNA-26b Targets NCEH1 in Underlying Subcutaneous Adipose Tissue  Louisa Cheung, Rachel M. Fisher, Natalia Kuzmina, Dongqing Li, Xi Li, Olivera Werngren, Lennart Blomqvist, Mona Ståhle, Ning Xu Landén  Journal of Investigative Dermatology  Volume 136, Issue 3, Pages 640-648 (March 2016) DOI: 10.1016/j.jid.2015.12.008 Copyright © 2015 The Authors Terms and Conditions

Figure 1 MicroRNA (miRNA) expression profiling of subcutaneous adipose tissue from patients with psoriasis. (a) miRNAs with a significant change in expression in subcutaneous adipose tissue underneath lesional skin (scAT-L) compared with scAT underneath nonlesional skin (scAT-NL) of psoriasis patients (n = 7). miRNAs with a fold change >1.3 and false discovery rate (q value) <5% are shown. (b) Unsupervised hierarchical clustering heat map illustrating the relative levels of these significantly differentially expressed miRNAs. Color intensity is scaled within each row so that the highest expression value corresponds to bright red and the lowest to bright green. (c) miRNA families and (d) miRNA clusters in which more than one family member was significantly regulated in scAT-L compared with scAT-NL. Journal of Investigative Dermatology 2016 136, 640-648DOI: (10.1016/j.jid.2015.12.008) Copyright © 2015 The Authors Terms and Conditions

Figure 2 Quantitative real-time polymerase chain reaction analysis of microRNAs (miRNAs) in subcutaneous adipose tissue from psoriasis patients. (a-i) Adipose tissue was collected underneath nonlesional skin (NL) and lesional skin (L) of psoriasis patients (n = 15). miRNA expression was normalized to the expression of U48 RNA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Wilcoxon matched pairs test. AU, arbitrary unit. Journal of Investigative Dermatology 2016 136, 640-648DOI: (10.1016/j.jid.2015.12.008) Copyright © 2015 The Authors Terms and Conditions

Figure 3 miR-26b directly targets NCEH1. (a) Nucleotide resolution of miR-26b binding sites in the 3′-untranslated region (3′UTR) of NCEH1 mRNA. Conserved regions: gray; miR-26b seed region: red; mutated miR-26b binding sites: small letters. (b) Cells were transfected with luciferase construct containing wild-type (WT) or mutant (MUT) NCEH1 3′UTR or empty vector, together with miR-26b precursor RNA (Pre–miR-26b) or control oligos (Pre–miR-Ctrl). Data are given as mean ± SD. **P < 0.01, Student t test. (c) Quantitative real-time PCR (QRT-PCR) analysis of NCEH1 in subcutaneous adipose tissue (scAT) underneath nonlesional skin (NL) and lesional skin (L). **P < 0.01, Wilcoxon matched pairs test. (d) Immunostaining of NCEH1 in scAT of psoriasis patients (n = 6). Sections were counterstained using 4′,6-diamidino-2-phenylindole (DAPI). Omission of the first antibody was used as negative control (Blank). Bars = 50 μm. Journal of Investigative Dermatology 2016 136, 640-648DOI: (10.1016/j.jid.2015.12.008) Copyright © 2015 The Authors Terms and Conditions

Figure 4 miR-26b regulates NCEH1 expression. (a) Quantitative real-time PCR (QRT-PCR) analysis of miR-26b and NCEH1 in adipocytes (n = 8), adipose tissue macrophages (n = 7), vascular endothelial cells (n = 3), fibroblasts (n = 3), T helper cells (Th; n = 1), regulatory T cells (Treg; n = 1) and cytotoxic T cells (Tc; n = 1). Data are given as mean ± SEM. *P < 0.05, one-way analysis of variance and Tukey post hoc test. (b) miR-26b and NCEH1 were analyzed in human peripheral blood monocytes (Mo) and Mo-derived macrophages (Mφ) by QRT-PCR and western blotting. *P < 0.05, **P < 0.01, Wilcoxon matched pairs test. miR-26b was overexpressed in (c) THP-1 cells, THP-1–derived macrophages, (d) preadipocytes, (e) vascular endothelial cells, and (f) fibroblasts. miR-26b and NCEH1 were analyzed by QRT-PCR and western blotting. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student t test. (b–f) QRT-PCR data given as mean ± SD. Journal of Investigative Dermatology 2016 136, 640-648DOI: (10.1016/j.jid.2015.12.008) Copyright © 2015 The Authors Terms and Conditions