Nobeyama Yoshimasa, Watanabe Yoshinori, Nakagawa Hidemi
Department of Dermatology, The Jikei University School of Medicine, Tokyo, Japan.
PLoS One. 2017 Oct 26;12(10):e0187047. doi: 10.1371/journal.pone.0187047. eCollection 2017.
Methylation of a CpG island (CGI; a dense cluster of CpGs) located in the 5' region of a gene suppresses that gene's transcription. The expression of G0/G1 switch gene 2 (G0S2) is potentially associated with tumorigenesis. The aim of this study is to elucidate the methylation status of the CGI located in the 5' region of G0S2 (hereinafter called 5' G0S2 CGI) in cutaneous squamous cell carcinoma (SCC).
Quantitative real-time methylation-specific PCR (RT-MSP) and bisulfite sequencing were performed to evaluate the methylation statuses of cutaneous SCC and normal epithelial cell samples. Quantitative real-time reverse transcription-PCR was performed to evaluate RNA expression levels. Immunohistochemical analysis was performed to detect protein expression.
G0S2 was suppressed in the five SCC cell lines with 5' G0S2 CGI methylation levels of nearly 100.0% and was expressed in the two normal cultured keratinocytes with methylation levels of almost 0.0%. G0S2 was re-expressed in SCC cell lines treated with a demethylating agent. The in vivo methylation levels of 5' G0S2 CGI as determined by RT-MSP varied widely (0.0% to 77.7%) in 17 cutaneous SCC samples and narrowly (0.1% to 7.3%) in 6 normal epidermis samples. Nine cutaneous SCC samples exhibited higher methylation levels than the highest methylation level (7.3%) of the 6 normal epidermis samples. Bisulfite sequencing showed dense methylated CpG sites within 5' G0S2 CGI in these highly methylated cutaneous SCC samples. The methylation levels of the cutaneous SCC samples did not correlate with any clinical parameters investigated or with histopathological grading.
G0S2 is silenced by aberrant DNA methylation in a subset of cutaneous SCCs.
位于基因5'区域的CpG岛（CGI；CpG的密集簇）甲基化会抑制该基因的转录。G0/G1开关基因2（G0S2）的表达可能与肿瘤发生有关。本研究的目的是阐明皮肤鳞状细胞癌（SCC）中位于G0S2 5'区域（以下称为5' G0S2 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，以评估皮肤SCC和正常上皮细胞样本的甲基化状态。进行定量实时逆转录PCR以评估RNA表达水平。进行免疫组织化学分析以检测蛋白质表达。
在5' G0S2 CGI甲基化水平接近100.0%的5种SCC细胞系中，G0S2被抑制，而在甲基化水平几乎为0.0%的2种正常培养角质形成细胞中表达。在用去甲基化剂处理的SCC细胞系中，G0S2重新表达。通过RT-MSP测定的17个皮肤SCC样本中5' G0S2 CGI的体内甲基化水平差异很大（0.0%至77.7%），而在6个正常表皮样本中差异较小（0.1%至7.3%）。9个皮肤SCC样本的甲基化水平高于6个正常表皮样本的最高甲基化水平（7.3%）。亚硫酸氢盐测序显示，在这些高度甲基化的皮肤SCC样本中，5' G0S2 CGI内存在密集的甲基化CpG位点。皮肤SCC样本的甲基化水平与所研究的任何临床参数或组织病理学分级均无相关性。
在一部分皮肤SCC中，G0S2因异常的DNA甲基化而沉默。
