MicroRNA Expression Profiling Identifies miR-31 and miR-485-3p as Regulators in the Pathogenesis of Discoid Cutaneous Lupus  Cristina Solé, Sandra Domingo, Berta Ferrer, Teresa Moliné, Josep Ordi-Ros, Josefina Cortés-Hernández  Journal of Investigative Dermatology  Volume 139, Issue 1, Pages 51-61 (January 2019) DOI: 10.1016/j.jid.2018.07.026 Copyright © 2018 The Authors Terms and Conditions

Figure 1 MicroRNA expression profiling in DLE compared with SCLE. (a) Heatmap from lesional skin biopsy samples from patients with DLE and SCLE illustrating levels of significantly changed microRNA expression (fold change >1.5, P < 0.01). (b) Expression of differentially expressed miRNAs was validated using quantitative real-time PCR in lesional skin of DLE (n = 20) and SCLE (n = 18) patients. The results for individual patients and mean are shown. Gene expression was normalized using U6 as endogenous control. Fold change in expression level was calculated using the 2–ΔΔCt method. (c) In situ hybridization illustrates miRNA dynamics in lesional and nonlesional DLE and SCLE samples and psoriasis. At least three biological replicates were performed; shown are representative images. Blue-purple color indicates miRNA expression. Scale bar = 2 mm. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005. DLE, discoid lupus erythematosus; miR, microRNA; SCLE, subacute cutaneous lupus erythematosus. Journal of Investigative Dermatology 2019 139, 51-61DOI: (10.1016/j.jid.2018.07.026) Copyright © 2018 The Authors Terms and Conditions

Figure 2 Expression of miR-31 and miR-485-3p after stimulatory conditions and during apoptosis. (a) Expression of miR-31 and miR-485-3p were analyzed in the cellular constituents of the skin including primary adult keratinocytes, dermal fibroblasts, and PBMCs using quantitative real-time PCR. (b) Expression of miR-31 and miR-485-3p in apoptotic primary keratinocytes and in transfected adult epidermal keratinocytes (HEKa cells) after UV and TGF-β1 stimulation. (c) BAX, BIM, p53, and caspase3 expression levels in apoptotic primary keratinocytes and in transfected adult epidermal keratinocytes (HEKa cells) after UV and TGF-β1 stimulation. Gene expression for miRNAs and mRNAs was normalized, respectively, using U6 or GADPH as endogenous control. Fold change in expression level was calculated using the 2–ΔΔCt method. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005. HD, healthy donors; Inh, inhibited; miR, microRNA; NS, not stimulated; Over, overexpressed; PBMC, peripheral blood mononuclear cell. Journal of Investigative Dermatology 2019 139, 51-61DOI: (10.1016/j.jid.2018.07.026) Copyright © 2018 The Authors Terms and Conditions

Figure 3 Overexpression of miRNA-31 increases the production of inflammatory cytokines by keratinocytes. UV or TGF-β1 stimulation of DLE keratinocytes and HEKa cells overexpressing miR-31 induces an increased expression and secretion of IL-8, IL-1β, IL-12, and TGF-β1 as measured by (a) qRT-PCR and (b) ELISA. (c) miR-31 regulates the NF-κB pathway in keratinocytes. Expression levels of NF-κB and of PPP6C and STK40, negative regulators, were measured by qRT-PCR in DLE keratinocytes and in transfected HEKa cells after UV and TGF-β1 stimulation. Gene expression was normalized using GADPH as endogenous gene control. Fold change in expression level was calculated using the 2–ΔΔCt method. (d) NF-κB expression was measured by immunofluorescence in overexpressed or inhibited miR-31 HEKa cells after 6 hours of treatment with TGF-β1. Scale bar = 20 μm. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005. DLE, discoid lupus erythematosus; HD, healthy donors; Inh, inhibited; miRNA, microRNA; Over, overexpressed; qRT-PCR, real-time quantitative reverse transcription–PCR; SCLE, subacute cutaneous lupus erythematosus. Journal of Investigative Dermatology 2019 139, 51-61DOI: (10.1016/j.jid.2018.07.026) Copyright © 2018 The Authors Terms and Conditions

Figure 4 Effect of miR-485-3p on expression of T-cell activation markers, T-cell differentiation, and fibrosis formation in primary fibroblast. Transfected miR-485-3p T cell stimulated with IL-1α or TGF-β1. (a) Flow cytometry diagram and percentages of CD69+CD4+ and CD69+CD8+. (b) qRT-PCR of T-cell activation genes and (c) transcription factor genes of Th1, Th2, and regulatory T cells and related cytokines. Overexpressed or inhibited miR-485-3p in fibroblast after stimulation with IL-1α or TGF-β1. (d) Gene expression analysis by qRT-PCR. (e) Immunofluorescence of TGF-βR1 protein levels. Scale bar = 20 μm. Data represent the mean ± standard error of the mean from three independent experiments. Genes were normalized to GADPH gene, and transfected controls were used for the fold changes. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005. Inh, inhibited; miR, microRNA; Over, overexpressed; qRT-PCR, real-time quantitative reverse transcription–PCR; Th, T helper. Journal of Investigative Dermatology 2019 139, 51-61DOI: (10.1016/j.jid.2018.07.026) Copyright © 2018 The Authors Terms and Conditions

Figure 5 miR-31 keratinocytes mediated neutrophil and monocyte migration and effect to their gene expression. PMNCs or primary PBMCs were placed into the upper of insert and transfected stimulated keratinocytes were in the bottom. (a) After 6 hours, the migrated primary neutrophil cells were counted. A representative image of the migrated primary neutrophil cells labeled with DAPI on the bottom of the Transwell membrane (Sarstedt, Nümbrecht, Germany) is shown. Percent migration of primary neutrophils relative to the transfected controls. Scale bar = 20 μm. (b) Migration of PBMCs were evaluated by flow cytometry. Representative dot blots indicating the percentages of migrated monocytes (nonclassical, intermediate, and classical) are shown. (c) Gene expression analysis by qRT-PCR were done for PMNC and PBMC co-culture with transfected stimulated keratinocytes. Comparisons were done by Student t test between overexpression and inhibition conditions. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005. Inh, inhibited; miR, microRNA; Over, overexpressed; PBMC, peripheral blood mononuclear cell; PMNC, neutrophil polymorphonuclear cell; qRT-PCR, real-time quantitative reverse transcription–PCR. Journal of Investigative Dermatology 2019 139, 51-61DOI: (10.1016/j.jid.2018.07.026) Copyright © 2018 The Authors Terms and Conditions

Figure 6 Proposed mechanism for miR-31 and miR-485-3p involvement in the pathogenesis of cutaneous lupus. (a) miR-31 overexpression in keratinocytes induced apoptosis and the production of inflammatory cytokines that in turn attract neutrophils/intermediate monocytes (IL-8, TGF-β1) and enhanced toward a Th1/regulatory T lymphocyte differentiation (IL-12, INFγ). Attracted neutrophils overexpressed IL-1β, CXCL9, and CXCL10, which could promote more leukocyte chemotaxis. Intermediate monocytes could be transformed into dendritic cells or macrophages to contribute to the maintenance of the inflammatory response. (b) Overexpression of miR-485-3p contributes to the inflammatory response by inducing T-cell activation by NF-κβ/PI3K1/PKCθ up-regulation. Overexpression of miR-485-3p in fibroblasts contributed to fibrosis formation by increasing levels of TGF-βR1, collagen (COL3A1), and smooth muscle actin (SMA). miR, microRNA; Th, T helper. Journal of Investigative Dermatology 2019 139, 51-61DOI: (10.1016/j.jid.2018.07.026) Copyright © 2018 The Authors Terms and Conditions