1. MicroRNA-203 Inversely Correlates with Differentiation Grade, Targets c-MYC, and Functions as a Tumor Suppressor in cSCC 
Warangkana Lohcharoenkal, Masako Harada, Jakob Lovén, Florian Meisgen, Ning Xu Landén, Lingyun Zhang, Jan Lapins, Kunal Das Mahapatra, Hao Shi, Liisa Nissinen, Veli-Matti Kähäri, Mona Ståhle, Enikö Sonkoly, Dan Grandér, Marie Arsenian-Henriksson, Andor Pivarcsi 
Journal of Investigative Dermatology 
Volume 136, Issue 12, Pages (December 2016)
DOI: /j.jid
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2. Figure 1
miR-203 is down-regulated in cSCCs. (a) Expression of epidermal differentiation markers (K10 and involucrin) and miR-203 was analyzed by immunofluorescence staining and in situ hybridization (ISH), respectively, in healthy human skin (n = 10) and squamous cell carcinomas (n = 40). Dashed lines demarcate tumor-stroma boundary. Scale bar = 100 μm. In situ hybridization score is shown as a box plot. (b) qPCR analysis of miR-203 in healthy skin (n = 13) and squamous cell carcinomas: grade I (n = 20), grade II (n = 20), and grade III (n = 15). (c) Expression level of miR-203 in cSCC cell lines (UT-SCC) compared with primary NHEKs. ∗P < 0.05, ∗∗∗P < 0.001, Student t test. cSCC, cutaneous squamous cell carcinoma; Epi, epidermis; K, keratin; miR, microRNA; NHEK, normal human epidermal keratinocytes; qPCR, quantitative PCR; Str, stroma; Tu, tumor.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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3. Figure 2
MiR-203 suppresses proliferation in cSCC cell lines. (a) Gene ontology term analysis of deregulated genes detected in miR-203–overexpressing UT-SCC-7 cells by Affymetrix whole transcript expression analysis (Affymetrix, Santa Clara, CA). (b) Heat map showing differentially expressed genes related to cell cycle and cell proliferation. Predicted target genes of miR-203 are labeled in red. (c) UT-SCC-7 and A431 were transfected with a synthetic precursor molecule for miR-203 (miR-203) or scrambled ODNs as negative control (scramble). After 48 hours, cell proliferation and cell cycle progression were measured by EdU labeling. (d) Expression of Ki67 proliferation marker in UT-SCC-7 and A431 after miR-203 transfection was determined by qPCR. The inlet showed immunofluorescence staining for Ki67. Scale bar = 50 μm. (e and f) Two- and three-dimensional (soft agar) colony formation assay of UT-SCC-7 and A431 transfected with miR-203 mimic. Optical density of crystal violet-stained colony is shown as bar chart. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, Student t test. cSCC, cutaneous squamous cell carcinoma; h, hours; miR, microRNA; ODN, scrambled oligonucleotide; qPCR, quantitative PCR; Scr, scramble.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

4. Figure 3
miR-203 suppresses migration and invasion of cSCC cells. (a) UT-SCC-7 and A431 transfected with miR-203 mimic or scrambled ODNs were subjected to scratch assay. Images were taken at 0, 7, 11, and 24 hours after the monolayer was scratched. Depicted are representative images and indications of the migration distances. ImageJ software (National Institutes of Health, Bethesda, MD) was used to determine the migration distance. Percentage of healing, which represents random cell migration, was calculated and is presented as a graph. (b and c) Transwell (Corning Life Sciences, New York, NY) migration and Matrigel (Corning Life Sciences) invasion assay of miR-203 mimic– or scrambled ODN–transfected UT-SCC-7 and A431. Data shown are means of cell counts obtained from three separate wells, and error bars represent standard deviations. ∗P < 0.05, ∗∗P < 0.01, Student t test. cSCC, cutaneous squamous cell carcinoma; h, hours; miR, microRNA; ODN, scrambled oligonucleotide.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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5. Figure 4
Inhibition of angiogenesis-inducing ability of cSCC by miR-203. (a) Heat map showing angiogenesis-related genes deregulated in miR-203–overexpressing cells. Putative miR-203 targets predicted by TargetScan algorithm ( are labeled in red. (b) qPCR analysis of VEGF-A, IL-8, and CCL2 mRNA levels in UT-SCC-7 and A431 cells transfected with miR-203 mimic or scramble oligo. (c) IL-8 level of supernatant collected from miR-203 mimic or scrambled ODNs transfected UT-SCC-7 and A431 cells detected by ELISA. (d) HUVECs tube formation assay was performed by resuspending HUVEC cells in supernatant collected from UT-SCC-7 and A431 cells transfected with miR-203 mimic or scrambled ODNs at 48 hours after transfection. HUVEC cell suspension was seeded into a 48-well plate with Matrigel (Corning Life Sciences, New York, NY) layer at the density of 4.5 × 104 per well. After 16–20 hours of incubation, photos were taken, and the number of well-organized nodes was counted. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, Student t test. h, hours; HUVEC, human umbilical vein endothelial cell; miR, microRNA; ODN, scrambled oligonucleotide; qPCR, quantitative PCR.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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6. Figure 5
MiR-203 directly targets c-MYC. (a) MetaCore (Thomson Reuters, New York, NY) analysis of genes regulated by miR-203 in miR-203–overexpressing cSCC cells. (b) c-MYC 3′UTR and MUT c-MYC 3′UTR reporter were constructed, and luciferase reporter assay was performed. (c) Western blot analysis of c-MYC and β-actin proteins in UT-SCC-7 cells transfected with miR-203 mimic or scrambled ODNs. (d) Expression level of c-MYC oncoprotein was analyzed in the section of healthy skin and cSCC samples by immunofluorescence staining. Scale bar = 200 μm. (e) c-MYC transcription factor activity of UT-SCC-7 cells transfected with miR-203 mimic was determined by TransAM c-MYC kit (Active Motif, Carlsbad, CA). (f) Expression level of proliferation-related MYC effectors in UT-SCC-7 cells transfected with miR-203 mimic or scrambled ODNs as determined by qPCR. (g) Restoration of c-MYC could override miR-203 effect on cell cycle. (h) Expression level of MYC and a well-known effector, CCND1, was measured in UT-SCC-7 double-transfected with miR-203/pcDNA or miR-203/pcDNA-MYC by qPCR. c-MYC protein level was determined by Western blot analysis. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, Student t test. A, adenine; C, cytosine; cSCC, cutaneous squamous cell carcinoma; Epi, epidermis; G, guanine; h, hours; miR, microRNA; MUT, mutated; ODN, scrambled oligonucleotide; PD, poorly differentiated squamous cell carcinoma; qPCR, quantitative PCR; Str, stroma; Tu, tumor; U, uracil; UTR, untranslated region; WD, well-differentiated squamous cell carcinoma; WT, wild type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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7. Figure 6
miR-203 suppresses tumor growth in xenograft model. (a) Stable miR-203 or scrambled ODNs overexpressing UT-SCC-7 cells was established. Ten million scramble or miR-203–overexpressing cells were injected into the left and right flank of each mouse, respectively (n = 6). (b) Tumor width and length were measured by caliper, and tumor volume was calculated as (width2 × length)/2. (c) When tumor volume reached 1,000 mm3 or mice conditions reached humane endpoint, tumors were harvested and weighed. Mean ± standard deviation for each group is indicated in the table. (d) Western blot analysis for the expression of MYC protein in tumors from scrambled ODNs or miR-203–overexpressing cells. (e) Immunofluorescent staining of c-MYC, c-Jun, and Ki67 in tumors. (f) Expression level of proliferation-related MYC effectors in the harvested tumors was determined by qPCR. (g) Immunohistochemical analysis of the endothelial marker CD36 (left) and quantification of the stained area (mean ± standard deviation of stained area) by ImagePro Plus software (Media Cybernetics, Rockville, MD) (right). Scale bar = 100 μm. ∗P < 0.05, ∗∗P < 0.01, Mann-Whitney U test. H&E, hematoxylin and eosin; miR, microRNA; ODN, scrambled oligonucleotide; qPCR, quantitative PCR; Scr, scramble; Wk, week.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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