Bilkent University, Department of Molecular Biology and Genetics, Ankara, Turkey.
BMC Cancer. 2011 Jun 6;11:223. doi: 10.1186/1471-2407-11-223.
Smad interacting protein-1 is a transcription factor that is implicated in transforming growth factor-β/bone morphogenetic protein signaling and a repressor of E-cadherin and human telomerase reverse transcriptase. It is also involved in epithelial-mesenchymal transition and tumorigenesis. However, genetic and epigenetic alterations of SIP1 have not been fully elucidated in cancers. In this study, we investigated mutations and promoter hypermethylation of the SIP1 gene in human hepatocellular carcinomas.
SIP1 expression was analyzed in HCC cell lines and primary tumors in comparison to normal and non-tumor liver tissues by using semi-quantitative RT-PCR, quantitative real-time RT-PCR and immunohistochemistry. Mutation and deletion screening of the SIP1 gene were performed by direct sequencing in HCC-derived cells. Restoration of SIP1 expression was sought by treating HCC cell lines with the DNA methyl transferase inhibitor, 5-AzaC, and the histone deacetylase inhibitor, TSA. SIP1 promoter methylation was analyzed by the combined bisulfite restriction analysis assay in in silico-predicted putative promoter and CpG island regions.
We found that the expression of SIP1 was completely lost or reduced in five of 14 (36%) HCC cell lines and 17 of 23 (74%) primary HCC tumors. Immunohistochemical analysis confirmed that SIP1 mRNA downregulation was associated with decreased expression of the SIP1 protein in HCC tissues (82.8%). No somatic mutation was observed in SIP1 exons in any of the 14 HCC cell lines. Combined treatment with DNA methyl transferase and histone deacetylase inhibitors synergistically restored SIP1 expression in SIP1-negative cell lines. Analysis of three putative gene regulatory regions revealed tumor-specific methylation in more than half of the HCC cases.
Epigenetic mechanisms contribute significantly to the downregulation of SIP1 expression in HCC. This finding adds a new level of complexity to the role of SIP1 in hepatocarcinogenesis.
Smad 相互作用蛋白-1 是一种转录因子，参与转化生长因子-β/骨形态发生蛋白信号转导，是 E-钙黏蛋白和人端粒酶逆转录酶的抑制剂。它还参与上皮-间充质转化和肿瘤发生。然而，SIP1 的遗传和表观遗传改变在癌症中尚未完全阐明。在这项研究中，我们研究了人肝细胞癌中 SIP1 基因的突变和启动子甲基化。
通过半定量 RT-PCR、实时定量 RT-PCR 和免疫组织化学，比较 HCC 细胞系和原发性肿瘤与正常和非肿瘤肝组织中 SIP1 的表达。在 HCC 衍生细胞中通过直接测序进行 SIP1 基因的突变和缺失筛选。通过用 DNA 甲基转移酶抑制剂 5-AzaC 和组蛋白去乙酰化酶抑制剂 TSA 处理 HCC 细胞系，寻求 SIP1 表达的恢复。通过在计算机预测的启动子和 CpG 岛区域的联合亚硫酸氢盐限制性分析测定法分析 SIP1 启动子甲基化。
我们发现，在 14 个 HCC 细胞系中的 5 个（36%）和 23 个原发性 HCC 肿瘤中的 17 个（74%）中，SIP1 的表达完全缺失或减少。免疫组织化学分析证实，SIP1mRNA 下调与 HCC 组织中 SIP1 蛋白表达减少相关（82.8%）。在任何 HCC 细胞系中均未观察到 SIP1 外显子的体细胞突变。DNA 甲基转移酶和组蛋白去乙酰化酶抑制剂联合治疗可协同恢复 SIP1 阴性细胞系中的 SIP1 表达。对三个假定的基因调控区域的分析表明，超过一半的 HCC 病例存在肿瘤特异性甲基化。
表观遗传机制显著促进了 HCC 中 SIP1 表达的下调。这一发现为 SIP1 在肝癌发生中的作用增添了新的复杂性。
