Tsai Ming-Hong, Chen Wen-Chi, Yu Sung-Liang, Chen Chun-Chieh, Jao Tzu-Ming, Huang Chi-Yen, Tzeng Sheng-Tai, Yen Sou-Jhy, Yang Ya-Chien
Department of Surgery, Cardinal Tien Hospital, New Taipei City, Taiwan.
School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan.
Ann Surg Oncol. 2015 Dec;22 Suppl 3:S1481-9. doi: 10.1245/s10434-015-4593-1. Epub 2015 May 13.
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.
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.
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.
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.
Shisa3是在肺癌中鉴定出的一种新型肿瘤抑制因子。然而，其在其他人类癌症中的抗肿瘤活性以及基因失活机制仍不清楚。
通过逆转录PCR（RT-PCR）和定量RT-PCR（RT-qPCR）检测SHISA3表达。通过亚硫酸氢盐测序和焦磷酸测序确定DNA甲基化。
在11种结直肠癌（CRC）细胞系中均观察到SHISA3表达下调，RT-qPCR在52例结直肠癌中的34例（65.4%）中进一步证实了这一点。6种CRC细胞系中的4种在用5-氮杂-2'-脱氧胞苷处理后可恢复SHISA3表达。通过亚硫酸氢盐测序鉴定了SHISA3第一内含子中5个CpG位点的肿瘤特异性甲基化，并通过亚硫酸氢盐焦磷酸测序在127对原发性CRC组织中对其甲基化水平进行了定量。肿瘤中SHISA3的甲基化水平明显高于其匹配的正常黏膜中的甲基化水平。此外，SHISA3高甲基化与II期和III期疾病患者疾病复发风险增加显著相关（P = 0.007），并且是CRC患者总生存期差[风险比（HR）2.9，95%置信区间（CI）1.5 - 5.8；P = 0.002]和无病生存期差（HR 4.0，95%CI 1.6 - 10.2；P = 0.003）的独立预测因子。
SHISA3基因在相当一部分CRC中发生表观遗传失活，其高甲基化在预测临床结果方面具有预后意义。所建立的定量亚硫酸氢盐焦磷酸测序测定法可能是一种经济有效的工具，可为CRC不良预后提供潜在的生物标志物。
