BACKGROUND Shisa3 is a novel tumor suppressor identified in lung cancer. However, its antitumor activity in other human cancers and the mechanism of gene inactivation remain unknown. METHODS SHISA3 expression was measured by reverse transcription-PCR (RT-PCR) and quantitative RT-PCR (RT-qPCR). DNA methylation was determined by bisulfite sequencing and pyrosequencing. RESULTS Down-regulation of SHISA3 expression was observed in all of 11 colorectal cancer (CRC) cell lines and was further confirmed in 34 (65.4 %) of 52 colorectal carcinomas by RT-qPCR. Four of six CRC cell lines could restore SHISA3 expression after treatment with 5-aza-2'-deoxycytidine. Tumor-specific methylation of five CpG sites in the first intron of SHISA3 was identified by bisulfite sequencing, and their methylation levels were quantified in 127 pairs of primary CRC tissues by bisulfite pyrosequencing. The methylation levels of SHISA3 in tumors were noticeably higher than that in their matched normal mucosae. In addition, SHISA3 hypermethylation was significantly associated with an increased risk of disease recurrence in patients with stage II and III disease (P = 0.007) and was an independent predictor of poor overall survival [hazard ratio (HR) 2.9, 95 % confidence interval (CI) 1.5-5.8; P = 0.002] and disease-free survival (HR 4.0, 95 % CI 1.6-10.2; P = 0.003) of CRC patients. CONCLUSIONS SHISA3 gene is epigenetically inactivated in a substantial fraction of CRC, and its hypermethylation is of prognostic significance in predicting clinical outcome. The quantitative bisulfite pyrosequencing assay established could be a cost-effective tool for providing a potential biomarker of adverse prognosis in CRC.

中文翻译：

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大肠癌中SHISA3的DNA超甲基化：不良预后的独立预测因子。

背景技术Shisa3是在肺癌中鉴定的新型肿瘤抑制剂。然而，其在其他人类癌症中的抗肿瘤活性和基因失活的机制仍然未知。方法通过逆转录PCR（RT-PCR）和定量RT-PCR（RT-qPCR）检测SHISA3的表达。通过亚硫酸氢盐测序和焦磷酸测序确定DNA甲基化。结果在所有11种结直肠癌（CRC）细胞系中均观察到SHISA3表达下调，并通过RT-qPCR在52种结直肠癌中的34种（65.4％）中进一步证实了这一点。6种CRC细胞系中的4种可以在用5-氮杂2'-脱氧胞苷处理后恢复SHISA3的表达。通过亚硫酸氢盐测序鉴定了SHISA3第一个内含子中五个CpG位点的肿瘤特异性甲基化，通过亚硫酸氢盐焦磷酸测序对127对原发性CRC组织中的甲基化水平进行定量。肿瘤中SHISA3的甲基化水平明显高于匹配的正常黏膜。此外，SHISA3甲基化水平高与II期和III期患者的疾病复发风险显着相关（P = 0.007），并且是整体生存不良的独立预测因子[危险比（HR）2.9，95％置信区间（ CI）1.5-5.8；P = 0.002]和CRC患者的无病生存期（HR 4.0，95％CI 1.6-10.2； P = 0.003）。结论SHISA3基因在大部分CRC中被表观遗传灭活，其高度甲基化对预测临床结局具有预后意义。