Nicastrin/miR-30a-3p/RAB31 Axis Regulates Keratinocyte Differentiation by Impairing EGFR Signaling in Familial Acne Inversa  Yanyan He, Haoxiang Xu, Chengrang Li, Xiaofeng Zhang, Pengjun Zhou, Xuemin Xiao, Wanlu Zhang, Yingda Wu, Rong Zeng, Baoxi Wang  Journal of Investigative Dermatology  Volume 139, Issue 1, Pages 124-134 (January 2019) DOI: 10.1016/j.jid.2018.07.020 Copyright © 2018 The Authors Terms and Conditions

Figure 1 miRNA profiling in familial AI patients with NCSTN mutations. (a) Skin sections from familial AI patients and healthy individuals were examined after hematoxylin and eosin staining. NCSTN mRNA and protein in familial AI patients and healthy individuals were detected using (a) immunohistochemical staining, (b) qRT-PCR, and (c) Western blot. (d) Cluster analysis of miRNAs induced or repressed in familial AI patients. (e) miRNA in familial AI patients compared with healthy individuals by qRT-PCR compared with microarray data (n = 5 per group). Scale bars = 100 μm. Values are expressed as the mean ± standard error of the mean of three individual experiments. ∗P < 0.05, ∗∗P < 0.01. AI, acne inversa; miR, microRNA; miRNA, microRNA; qRT-PCR, quantitative real-time reverse transcriptase–PCR. Journal of Investigative Dermatology 2019 139, 124-134DOI: (10.1016/j.jid.2018.07.020) Copyright © 2018 The Authors Terms and Conditions

Figure 2 Cluster analysis of aberrant miRNA expression in NcstnΔKCmice. (a, b) Schematic overview of the establishment of NcstnΔKC mice and sample collection. (c) Representative images of normal and NcstnΔKC mice harvested after tamoxifen injection. (d) Body weight changes and skin sections from normal and NcstnΔKC mice were examined after hematoxylin and eosin or stained with antibodies against (e) CD45 or (h) NCSTN/KRT14. (f, g) NCSTN mRNA and protein were examined by qRT-PCR and Western blot. (i) Cluster analysis of miRNA between normal and NcstnΔKC mice. (j) miRNAs in normal and NcstnΔKC mice determined by qRT-PCR compared with microarray data (six pairs [qRT-PCR] and 4 pairs [microarray]). Scale bars = 100 μm (H&E) or 25 μm (immunofluorescence). Values are expressed as the mean ± standard error of the mean of three individual experiments. ∗∗∗P < 0.001. H&E, hematoxylin and eosin; KRT, keratin; miRNA, microRNA; qRT-PCR, quantitative real-time reverse transcriptase–PCR. Journal of Investigative Dermatology 2019 139, 124-134DOI: (10.1016/j.jid.2018.07.020) Copyright © 2018 The Authors Terms and Conditions

Figure 3 Defective NCSTN expression down-regulated miR-30a-3p in keratinocytes. (a, b) Immunofluorescence with KRT 14 and in situ hybridization for miR-30a-3p were performed on skin sections from familial AI patients and NcstnΔKC mice. Scale bar = 100 μm (original magnification ×100) or 25 μm (original magnification ×600). (c) qRT-PCR of miR-30a-3p, miR-100-5p, and GATA3 in NCSTN-siRNA–treated HaCaT cells. (d, e) GATA3 mRNA and protein in AI patients were detected. Scale bar = 50 μm. (f) The miR-30a promoter regions enriched by GATA3 antibody-mediated ChIP were amplified by PCR and run on a 2% agarose gel. (g) The luciferase activity in HaCaT cells co-transferred with reporter carrying miR-30a promoter with wild-type (WT) or mutated (MUT) GATA3-binding sites was detected 48 hours after transfection. (h) qRT-PCR of GATA3 in NCSTN-siRNA& GV147-GATA3-plasmid–treated HaCaT cells. Values are expressed as the mean ± standard error of the mean of three individual experiments. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. bp, base pair; ChIP, chromatin immunoprecipitation; KRT, keratin; qRT-PCR, quantitative real-time reverse transcriptase–PCR; miR, microRNA; siRNA, small interfering RNA. Journal of Investigative Dermatology 2019 139, 124-134DOI: (10.1016/j.jid.2018.07.020) Copyright © 2018 The Authors Terms and Conditions

Figure 4 RAB31 is down-regulated by miR-30a-3p. (a) Western blot of RAB31 in HaCaT cells and primary mouse keratinocytes after pre/anti-miR-30a-3p or pre/anti-scramble transfection. (b–d) RAB31 in familial AI patients (n = 5 per group) and NcstnΔKC mice (n = 6 mice per group, representative images are shown) was examined by (b, c) Western blot and (d) immunofluorescence. Scale bars = 12.5 μm (human) or 25 μm (mice). (e) Affinity purification products with biotin-miR-30a-3p from HaCaT cells were amplified by PCR and run on a 2% agarose gel. (f) Luciferase activities containing the full-length 3′ UTRs of human or mouse RAB31 in HaCaT cells and primary mouse keratinocytes after pre/anti-miR-30a-3p or pre/anti-scramble transfection. Values are expressed as the mean ± standard error of the mean of three individual experiments. ∗P < 0.05, ∗∗P < 0.01. AI, acne inversa; bp, base pair; miR, microRNA; NS, no significant change; UTR, untranslated region; WT, wild type. Journal of Investigative Dermatology 2019 139, 124-134DOI: (10.1016/j.jid.2018.07.020) Copyright © 2018 The Authors Terms and Conditions

Figure 5 NCSTN knockdown blocks EGFR signaling through miR-30a-3p/RAB31. RAB31 level was examined in HaCaT cells co-transfected with (a) NCSTN-siRNA&pre-miR-30a-3p or (d) anti-miR-30a-3p&RAB31-siRNA 48 hours after transfection. The treated cells were serum-starved overnight and stimulated with EGF for 5 minutes, then replaced with complete culture medium. Cells were (b, e) stained with EGFR antibodies or lysed for (c, f) Western blot at indicated time points. (g) Immunofluorescence and Western blot for p-EGFR and total EGFR in skin lesions of (h) familial AI patients (n = 5 per group) and (i) NcstnΔKC mice (n = 6 mice per group; representative images are shown). Scale bars = 10 μm in b and e or 50 μm in g. Values are expressed as the mean ± standard error of the mean of three individual experiments. *P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. AI, acne inversa; min, minute; miR, microRNA; p-, phosphorylated; siRNA, small interfering RNA. Journal of Investigative Dermatology 2019 139, 124-134DOI: (10.1016/j.jid.2018.07.020) Copyright © 2018 The Authors Terms and Conditions

Figure 6 Defective EGFR relating to NCSTN leads to abnormal differentiation of keratinocytes. (a) Analysis of keratin 1 and loricrin in HaCaT cells co-transfected with NCSTN-siRNA&pre-miR-30a-3p or anti-miR-30a-3p&RAB31-siRNA, then stimulated with EGF and collected for Western blot 24 hours after stimulation. (b) Immunofluorescence of keratin 1 and loricrin in skin lesions of familial AI patients and NcstnΔKC mice. (c) Western blot of keratin 1 and loricrin in skin lesions from familial AI patients (five in each group, representative images are shown) and NcstnΔKC mice (six in each group, representative images are shown). Scale bars = 12.5 μm (human) or 25 μm (mice). Values are expressed as the mean ± standard error of the mean of three individual experiments. *P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. AI, acne inversa; siRNA, small interfering RNA. Journal of Investigative Dermatology 2019 139, 124-134DOI: (10.1016/j.jid.2018.07.020) Copyright © 2018 The Authors Terms and Conditions