Gene Expression and Methylation Analysis in Melanomas and Melanocytes From the Same Patient: Loss of NPM2 Expression Is a Potential Immunohistochemical Marker for Melanoma
Gene Expression and Methylation Analysis in Melanomas and Melanocytes From the Same Patient: Loss of NPM2 Expression Is a Potential Immunohistochemical Marker for Melanoma
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DOI：
10.3389/fonc.2018.00675
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发表时间：
2019-01-21
影响因子：
4.7
通讯作者：
Nishigori, Chikako
中科院分区：
文献类型：
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作者：
Fujiwara, Susumu;Nagai, Hiroshi;Nishigori, Chikako
DNA甲基化被认为是恶性黑色素瘤发展的主要表观遗传机制。由于 DNA 甲基化可能受到环境因素的影响，因此最好比较来自同一患者的癌细胞和正常细胞。为了比较同一个体的黑色素瘤组织和黑色素细胞的甲基化状态，我们采用了一种新型表皮片培养技术，从黑色素瘤患者未受影响的部位分离正常黑色素细胞。我们还分析了原发性和转移性黑色素瘤样本、三种市售黑色素细胞和四种黑色素瘤细胞系。 DNA甲基化数据的聚类分析将新分离的黑色素瘤和黑色素细胞归为同一组，而四种黑色素瘤细胞系聚集在一个遥远的进化枝中。此外，除了先前研究中确定的基因座（COL1A2、GPX3）之外，我们的分析还发现了几个新基因座（KRTCAP3、AGAP2、ZNF490）的甲基化；然而，在新鲜的黑色素瘤样本中没有观察到后两者。随后的研究表明，NPM2在黑色素瘤中高度甲基化并下调，这与之前的报道一致。在许多正常黑色素细胞中，NPM2 显示出明显的免疫组织化学染色，而其表达在恶性黑色素瘤细胞中缺失。特别是，恶性黑色素瘤的上皮内病变是临床实践中的一个重要挑战，可以将其与良性痣区分开。目前的研究结果表明在表观遗传学研究中使用新鲜黑色素瘤样本而不是黑色素瘤细胞系和黑色素细胞的重要性。此外，NPM2 免疫反应性可用于区分黑色素瘤与正常黑色素细胞或良性疾病。
DNA methylation is considered the primary epigenetic mechanism underlying the development of malignant melanoma. Since DNA methylation can be influenced by environmental factors, it is preferable to compare cancer and normal cells from the same patient. In order to compare the methylation status in melanoma tissues and melanocytes from the same individuals, we employed a novel epidermal sheet cultivation technique to isolate normal melanocytes from unaffected sites of melanoma patients. We also analyzed primary and metastatic melanoma samples, three commercially available melanocytes, and four melanoma cell lines. Cluster analysis of DNA methylation data classified freshly isolated melanomas and melanocytes into the same group, whereas the four melanoma cell lines were clustered together in a distant clade. Moreover, our analysis discovered methylation at several novel loci (KRTCAP3, AGAP2, ZNF490), in addition to those identified in previous studies (COL1A2, GPX3); however, the latter two were not observed in fresh melanoma samples. Subsequent studies revealed that NPM2 was hypermethylated and downregulated in melanomas, which was consistent with previous reports. In many normal melanocytes, NPM2 showed distinct immunohistochemical staining, while its expression was lost in malignant melanoma cells. In particular, intraepithelial lesions of malignant melanoma, an important challenge in clinical practice, could be distinguished from benign nevi. The present findings indicate the importance of using fresh melanoma samples, not melanoma cell lines and melanocytes in epigenetic studies. In addition, NPM2 immunoreactivity could be used to differentiate melanomas from normal melanocytes or benign disease.