Nobeyama Yoshimasa, Nakagawa Hidemi
Department of Dermatology, The Jikei University School of Medicine, Minato-ku, Tokyo, Japan.
PLoS One. 2017 Sep 6;12(9):e0184444. doi: 10.1371/journal.pone.0184444. eCollection 2017.
Methylation of a CpG island (CGI; a dense cluster of CpGs) located in the 5' region of a gene suppresses transcription of that gene. Interferon regulatory factor 6 (IRF6) is associated with the expression of interferon, which is used as an effective adjuvant therapy for melanoma, and is regarded as a tumor suppressor. However, little is known about the methylation status of the IRF6 gene in melanoma.
The purpose was to determine the methylation status of the CGI located in the 5' region of IRF6 (5' IRF6 CGI) in melanoma.
Quantitative real-time methylation-specific PCR (RT-MSP) and bisulfite sequencing were performed to examine IRF6 gene methylation status. Quantitative real-time reverse transcription-PCR (RT-PCR) was performed to examine IRF6 expression.
The methylation level of the 5' IRF6 CGI was completely inversely correlated with cell sensitivity to interferon-β in eight examined melanoma cell lines. These methylation levels were high in the melanoma cell lines with suppression of IRF6 expression and were low in the cell lines with IRF6 expression. The methylation levels of the 5' IRF6 CGI ranged widely from 0.0% to 65.4% in 21 clinical melanoma samples but showed a narrow range of low levels between 0.0% to 7.2% in 24 clinical melanocytic nevus samples. These methylation levels were not associated with clinical parameters except for melanoma subtypes.
IRF6 is aberrantly silenced by DNA methylation of the 5' IRF6 CGI in melanoma. The methylation status of IRF6 is potentially associated with the sensitivity of melanoma to interferon.
位于基因5'区域的CpG岛（CGI；CpG的密集簇）的甲基化会抑制该基因的转录。干扰素调节因子6（IRF6）与干扰素的表达相关，干扰素用作黑色素瘤的有效辅助治疗，并且被视为一种肿瘤抑制因子。然而，关于黑色素瘤中IRF6基因的甲基化状态知之甚少。
确定黑色素瘤中位于IRF6基因5'区域（5' IRF6 CGI）的CGI的甲基化状态。
进行定量实时甲基化特异性PCR（RT-MSP）和亚硫酸氢盐测序以检测IRF6基因的甲基化状态。进行定量实时逆转录PCR（RT-PCR）以检测IRF6的表达。
在8个检测的黑色素瘤细胞系中，5' IRF6 CGI的甲基化水平与细胞对干扰素-β的敏感性完全呈负相关。在IRF6表达受抑制的黑色素瘤细胞系中这些甲基化水平较高，而在具有IRF6表达的细胞系中则较低。在21个临床黑色素瘤样本中，5' IRF6 CGI的甲基化水平范围广泛，从0.0%至65.4%，但在24个临床黑素细胞痣样本中显示出较窄的低水平范围，介于0.0%至7.2%之间。除黑色素瘤亚型外，这些甲基化水平与临床参数无关。
在黑色素瘤中，IRF6因5' IRF6 CGI的DNA甲基化而异常沉默。IRF6的甲基化状态可能与黑色素瘤对干扰素的敏感性相关。
