Guo Yan-Li, Shan Bao-En, Guo Wei, Dong Zhi-Ming, Zhou Zhen, Shen Su-Peng, Guo Xin, Liang Jia, Kuang Gang
Laboratory of Pathology, Hebei Cancer Institute, The Fourth Hospital of Hebei Medical University, Jiankanglu 12, Shijiazhuang, 050011, Hebei, China.
J Biomed Sci. 2017 Jan 11;24(1):6. doi: 10.1186/s12929-016-0308-6.
The DACT (Dishevelled-associated antagonist of β-catenin) family of scaffold proteins may play important roles in tumorigenesis. However, the epigenetic changes of DACT1, 2, 3 and their effect on esophageal squamous cell carcinoma (ESCC) have not been investigated so far. The aim of this study was to investigate the promoter methylation and expression of DACT family, in order to elucidate more information on the role of DACT with regard to the progression and prognosis of ESCC.
MSP and BGS methods were respectively applied to examine the methylation status of DACT; RT-PCR, Western blot and immunohistochemistry methods were respectively used to determine the mRNA and protein expression of DACT; MTT, Colony-formation and Wound-healing assay were performed to assess the effect of DACT1 and DACT2 on proliferation and migration of esophageal cancer cells.
Frequent reduced expression of DACT1, DACT2 and DACT3 were found in esophageal cancer cell lines and the expression levels of DACT1 and DACT2 were reversed by 5-Aza-Dc. Decreased mRNA and protein expression of DACT1 and DACT2 were observed in ESCC tumor tissues and were associated with the methylation status of transcription start site (TSS) region. The hypermethylation of CpG islands (CGI) shore region in DACT1 was observed both in tumor and corresponding adjacent tissues but wasn't related to the transcriptional inhibition of DACT1. The methylation status of TSS region in DACT1 and DACT2 and the protein expression of DACT2 were independently associated with ESCC patients' prognosis.
The TSS region hypermethylation may be one of the main mechanisms for reduced expression of DACT1 and DACT2 in ESCC. The simultaneous methylation of DACT1 and DACT2 may play important roles in progression of ESCC and may serve as prognostic methylation biomarkers for ESCC patients.
支架蛋白DACT（β-连环蛋白相关的Dishevelled拮抗剂）家族可能在肿瘤发生中发挥重要作用。然而，迄今为止，尚未研究DACT1、2、3的表观遗传变化及其对食管鳞状细胞癌（ESCC）的影响。本研究旨在探讨DACT家族的启动子甲基化和表达情况，以阐明DACT在ESCC进展和预后方面作用的更多信息。
分别采用甲基化特异性PCR（MSP）和亚硫酸氢盐基因组测序（BGS）方法检测DACT的甲基化状态；分别使用逆转录-聚合酶链反应（RT-PCR）、蛋白质免疫印迹法（Western blot）和免疫组织化学方法测定DACT的mRNA和蛋白质表达；进行MTT法、集落形成实验和伤口愈合实验，以评估DACT1和DACT2对食管癌细胞增殖和迁移的影响。
在食管癌细胞系中发现DACT1、DACT2和DACT3的表达经常降低，5-氮杂-2'-脱氧胞苷（5-Aza-Dc）可逆转DACT1和DACT2的表达水平。在ESCC肿瘤组织中观察到DACT1和DACT2的mRNA和蛋白质表达降低，且与转录起始位点（TSS）区域的甲基化状态相关。在肿瘤组织及其相应的癌旁组织中均观察到DACT1中CpG岛（CGI）岸区的高甲基化，但这与DACT1的转录抑制无关。DACT1和DACT2中TSS区域的甲基化状态以及DACT2的蛋白质表达与ESCC患者的预后独立相关。
TSS区域高甲基化可能是ESCC中DACT1和DACT2表达降低的主要机制之一。DACT1和DACT2的同时甲基化可能在ESCC进展中起重要作用，并可能作为ESCC患者的预后甲基化生物标志物。
