methylation analysis in melanomas and melanocytes from the same patient. methylation analysis in melanomas and melanocytes from the same patient

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. Here, we employed a novel epidermal sheet cultivation technique to isolate normal melanocytes from unaffected sites of melanoma patients and compared the methylation status with melanoma tissues from the same individuals. 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 and indicated that NPM2 immunoreactivity could be used to differentiate melanomas from normal melanocytes or benign disease. Our results highlight the importance of using matched fresh melanoma and melanocyte samples in epigenetic studies. Illumina Infinium 450k Human Methylation Beadchip Overall design: total 19 samples, 8 melanocytes and 11 melanomas.methylation were performed on 5 cultured melanocytes, 7 melanomas (giving 4 matched donor pairs), 4 commercial melanoma cell lines and 3 commercial melanocyte cell lines.

DNA甲基化（DNA methylation）被认为是恶性黑色素瘤发生发展的主要表观遗传机制。由于DNA甲基化可受环境因素影响，因此优先选取同一患者的癌组织与正常组织进行比对分析。本研究采用一种新型表皮片层培养技术，从黑色素瘤患者的未受累部位分离正常黑素细胞，并将其与同一患者的黑色素瘤组织的甲基化状态进行对比。

我们还对原发性与转移性黑色素瘤样本、3株商用黑素细胞系以及4株黑色素瘤细胞系进行了分析。对DNA甲基化数据的聚类分析结果显示，新鲜分离的黑色素瘤与黑素细胞被归为同一类群，而4株黑色素瘤细胞系则聚为一个独立的进化分支。

此外，本研究还发现了数个新的甲基化位点（KRTCAP3、AGAP2、ZNF490），同时涵盖此前研究中已报道的位点（COL1A2、GPX3）；但后两个位点并未在新鲜黑色素瘤样本中被检测到。

后续研究表明，NPM2在黑色素瘤中呈高甲基化状态且表达下调，这与此前的研究报道一致，同时提示NPM2免疫反应性可用于区分黑色素瘤与正常黑素细胞或良性病变。本研究结果凸显了在表观遗传研究中使用匹配的新鲜黑色素瘤与黑素细胞样本的重要性。

Illumina Infinium 450k人类甲基化微珠芯片
实验设计：共纳入19份样本，其中8份黑素细胞样本与11份黑色素瘤样本。甲基化检测覆盖5株培养黑素细胞、7份黑色素瘤组织（对应4对匹配供体样本）、4株商用黑色素瘤细胞系以及3株商用黑素细胞系。