*Asbestos Diseases Research Institute, University of Sydney, Sydney, Australia; †Department of Anatomical Pathology, Flinders Medical Centre, Adelaide, Australia; ‡Cardiothoracic Surgical Unit, Royal Prince Alfred Hospital; The Baird Institute, Sydney Medical School, University of Sydney, Sydney, Australia; and §Biomedical Research Center, Sir Runrun Shaw Hospital, Zhejiang University, Hangzhou, China.
J Thorac Oncol. 2013 Oct;8(10):1317-28. doi: 10.1097/JTO.0b013e3182a0840a.
INTRODUCTION: Epigenetic inactivation of tumor suppressor genes is involved in the development of malignant pleural mesothelioma (MPM). ZIC1, a potential tumor suppressor gene involved in regulating cell growth and apoptosis, was investigated in MPM cell lines and tumors. METHODS: ZIC1 expression and promoter methylation were evaluated in MPM cell lines and tumor samples by quantitative polymerase chain reaction (PCR), Combined Bisulfite Restriction Analysis, and methylation-specific PCR. ZIC1 was reexpressed in cell lines and functional effects were assessed. miRNA expression was quantified by microarray and reverse transcription quantitative PCR. ZIC1 knockdown and miRNA inhibitors were used to study the relationship between ZIC1 and miRNA expression and confirmed by chromatin immunoprecipitation PCR. RESULTS: ZIC1 expression was low in MPM cells, and was correlated with ZIC1 promoter methylation and reversed upon decitabine treatment. ZIC1 reexpression inhibited proliferation and invasion in MPM cells whereas knockdown enhanced the growth of MeT-5A. In MPM tumor samples ZIC1 expression was either low or undetectable, with promoter methylation observed in 16 of 24 cases. The overexpression of miR-23a and miR-27a was reduced by ZIC1 reexpression, with inhibitors of miR-23a or miR-27a reducing colony formation. miR-23a overexpression was also associated with shorter survival of MPM patients. CONCLUSION: ZIC1 is down-regulated in MPM through promoter methylation and acts as a tumor suppressor through down-regulation of its direct targets miR-23a and miR-27a.
简介：肿瘤抑制基因的表观遗传失活参与了恶性胸膜间皮瘤（MPM）的发展。ZIC1 是一个潜在的肿瘤抑制基因，参与调节细胞生长和凋亡，在 MPM 细胞系和肿瘤中进行了研究。
方法：通过定量聚合酶链反应（PCR）、联合亚硫酸氢盐限制性分析和甲基化特异性 PCR，评估 ZIC1 在 MPM 细胞系和肿瘤样本中的表达和启动子甲基化。在细胞系中重新表达 ZIC1，并评估其功能影响。通过微阵列和逆转录定量 PCR 定量 miRNA 表达。使用 ZIC1 敲低和 miRNA 抑制剂来研究 ZIC1 和 miRNA 表达之间的关系，并通过染色质免疫沉淀 PCR 进行验证。
结果：ZIC1 在 MPM 细胞中的表达较低，与 ZIC1 启动子甲基化相关，并在去甲基化药物处理后逆转。ZIC1 再表达抑制了 MPM 细胞的增殖和侵袭，而 ZIC1 敲低则增强了 MeT-5A 的生长。在 MPM 肿瘤样本中，ZIC1 的表达要么较低，要么无法检测到，在 24 例中有 16 例观察到启动子甲基化。ZIC1 再表达降低了 miR-23a 和 miR-27a 的过表达，miR-23a 或 miR-27a 的抑制剂减少了集落形成。miR-23a 的过表达也与 MPM 患者的生存时间较短有关。
结论：ZIC1 通过启动子甲基化在 MPM 中下调，并通过下调其直接靶标 miR-23a 和 miR-27a 作为肿瘤抑制因子发挥作用。
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