Silencing of G0/G1 switch gene 2 in cutaneous squamous cell carcinoma

BackgroundMethylation 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).MethodsQuantitative 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.ResultsG0S2 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.ConclusionsG0S2 is silenced by aberrant DNA methylation in a subset of cutaneous SCCs.背景：位于基因5'端区域的CpG岛（CpG island, CGI，即CpG二核苷酸密集簇）的甲基化会抑制该基因的转录。G0/G1转换基因2（G0/G1 switch gene 2, G0S2）的表达与肿瘤发生存在潜在关联。本研究旨在明确皮肤鳞状细胞癌（cutaneous squamous cell carcinoma, SCC）中G0S2基因5'端区域CpG岛（下文简称5' G0S2 CGI）的甲基化状态。 方法：采用实时荧光定量甲基化特异性PCR（quantitative real-time methylation-specific PCR, RT-MSP）与亚硫酸氢盐测序，对皮肤鳞状细胞癌及正常上皮细胞样本的甲基化状态进行评估；采用实时荧光定量反转录PCR检测RNA表达水平；采用免疫组化分析检测蛋白表达情况。 结果：在5' G0S2 CGI甲基化水平接近100.0%的5株皮肤鳞状细胞癌细胞系中，G0S2表达被抑制；而在甲基化水平几乎为0.0%的2株正常培养角质形成细胞中，G0S2呈表达状态。经去甲基化试剂处理后，皮肤鳞状细胞癌细胞系可重新表达G0S2。通过RT-MSP检测发现，17例皮肤鳞状细胞癌样本的体内5' G0S2 CGI甲基化水平分布范围较广（0.0%~77.7%），6例正常表皮样本的甲基化水平分布范围较窄（0.1%~7.3%）。其中9例皮肤鳞状细胞癌样本的甲基化水平高于6例正常表皮样本的最高甲基化水平（7.3%）。亚硫酸氢盐测序结果显示，在这些高甲基化的皮肤鳞状细胞癌样本中，5' G0S2 CGI内存在密集的甲基化CpG位点。皮肤鳞状细胞癌样本的甲基化水平与所研究的各项临床参数及组织病理学分级均无相关性。 结论：在部分皮肤鳞状细胞癌中，G0S2基因会因异常DNA甲基化而发生沉默。