Serum miR-16: A Potential Biomarker for Predicting Melanoma Prognosis

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Serum miR-16: A Potential Biomarker for Predicting Melanoma Prognosis Sen Guo, Weinan Guo, Shuli Li, Wei Dai, Nan Zhang, Tao Zhao, Huina Wang, Jingjing Ma, Xiuli Yi, Rui Ge, Gang Wang, Tianwen Gao, Chunying Li Journal of Investigative Dermatology Volume 136, Issue 5, Pages 985-993 (May 2016) DOI: 10.1016/j.jid.2015.12.041 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Significant reduction of serum miR-16 level in melanoma patients. (a) Levels of serum miR-16 in 120 melanoma patients and 120 cancer-free controls. miR-16 level was normalized to Cel-miR-39 in serum. (b) Generated receiver operating characteristic curve of serum miR-16 had an area under the curve (AUC) value of 0.779. Using a cutoff value of 0.585, sensitivity and specificity were 80.0% and 71.7%, respectively, in distinguishing melanoma patients from cancer-free controls. Two-tailed Student t test was used to analyze significant differences. ***P < 0.001. Journal of Investigative Dermatology 2016 136, 985-993DOI: (10.1016/j.jid.2015.12.041) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Association between melanoma American Joint Committee on Cancer (AJCC) stages and miR-16 levels both in sera and tissues. (a) Serum miR-16 levels of melanoma patients at different AJCC stages. (b) Correlation between serum miR-16 levels and melanoma AJCC stages was tested by Spearman’s rank correlation analysis, with r and P-values indicated. (c) Expression levels of miR-16 in 120 melanoma tissues and 120 matched normal nevus tissues. (d) Tissue miR-16 levels of melanoma patients at different AJCC stages. (e) Correlation between tissue miR-16 levels and melanoma AJCC stages was tested by Spearman’s rank correlation analysis, with r and P-values indicated. Two-tailed Student’s t test was used to analyze significant differences. *P < 0.05, **P < 0.01, ***P < 0.001. Journal of Investigative Dermatology 2016 136, 985-993DOI: (10.1016/j.jid.2015.12.041) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Significant correlation between serum miR-16 level and tissue Ki-67 expression. (a–e) Representative immunohistochemical staining of Ki-67 (red). (a) Nevus. (b) Stage I melanoma. (c) Stage II melanoma. (d) Stage III melanoma. (e) Stage IV melanoma. Bar = 600 μm. (f) Ki-67 expression staining score in melanoma tissues at different American Joint Committee on Cancer stages and in nevus tissues. (g) Serum miR-16 levels in different Ki-67 expression groups. (h) Correlation between serum miR-16 levels and Ki-67 expressions tested by Spearman rank correlation analysis. (i) Tissue miR-16 levels in different Ki-67 expression groups. (j) Correlation between tissue miR-16 levels and Ki-67 expressions tested by Spearman rank correlation analysis. Two-tailed Student t test was used to analyze significant differences. *P < 0.05; **P < 0.01; ***P < 0.001. Journal of Investigative Dermatology 2016 136, 985-993DOI: (10.1016/j.jid.2015.12.041) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Kaplan–Meier analysis for overall survival of melanoma patients. Kaplan-Meier survival curves plotted for melanoma patients (a) on serum miR-16 level only (P < 0.0001); (b) on serum miR-16 levels with age ≤50 years (P = 0.2231) or >50 years (P = 0.0008); (c) on serum miR-16 levels with male (P = 0.0045) or female (P = 0.0382); (d) on serum miR-16 levels at American Joint Committee on Cancer (AJCC) stages I and II (P = 0.8536) or AJCC stages III or IV (P = 0.0004); (e) on serum miR-16 levels with tumor thickness ≤1 mm (P = 0.7411) or >1 mm (P = 0.0228); (f) on serum miR-16 levels with (P = 0.0100) or without ulceration (P = 0.3306); (g) on serum miR-16 levels with sun-exposed (P = 0.0006) or protected sites (P = 0.2357). Journal of Investigative Dermatology 2016 136, 985-993DOI: (10.1016/j.jid.2015.12.041) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Suppressive role of miR-16 in melanoma development. (a) WM793B, 451LU, and A2058 melanoma cell lines were transfected with miR-16 expression vector or negative control. miR-16 expression was evaluated 48 hours later by quantitative real-time PCR. (b–d) Apoptotic cells, cell viability, and cell cycle distribution were analyzed in melanoma cell lines 48 hours after transfection with miR-16 or NC. (e–g) Comparison of tumor engraftment sizes between miR-NC transfected A2058 cells and miR-16 transfected cells 40 days after injection into nude mice. (h) Comparison of intratumoral Ki-67 expression between miR-16 transfected engraftment and miR-NC transfected engraftment 40 days after injection. Bar = 20 μm. (i) Candidate targets of miR-16 were shown. Two-tailed Student t test was used to analyze significant differences. *P < 0.05; **P < 0.01; ***P < 0.001. DAPI, 4',6-diamidino-2-phenylindole. Journal of Investigative Dermatology 2016 136, 985-993DOI: (10.1016/j.jid.2015.12.041) Copyright © 2016 The Authors Terms and Conditions