METHODS: Serum samples from 90 HCC patients and 27 healthy donors were collected in this study. The methylation-specific PCR (MSP) was performed to evaluate promoter methylation status of APC gene. RT-qPCR was used to detect the mRNA expression of APC, β-Catenin, c-Myc and Cyclin D1, meanwhile the protein expression were analyzed by Western blot.
RESULTS: The positive rate of APC gene methylation in HCC patients (46.67%) was higher than healthy donors (11.11%). APC gene exhibited marked hypermethylation in the patients of TNM III-IV stage when compared to the patients of TNM I-II stage , the methylation status of APC gene was correlated with tumor size and lymph node metastasis whereas the APC gene methylation showed no relationship with the patient\'s sex and age. APC methylation may be associated with APC expression level, APC expression in HCC cells is silenced by aberrant promoter hypermethylation. In HCC patients with methylated APC, the mRNA and protein expression of β-Catenin, c-Myc and Cyclin D1 were higher than the unmethylated patient subgroup and healthy donors.
CONCLUSIONS: The downregulation of APC in HCC samples was associated with promoter hypermethylation. APC methylation could be used as a novel diagnostic biomarker in HCC, which was associated with regulation of Wnt/β-Catenin signal pathway.
方法:本研究收集了90例HCC患者和27例健康供体的血清样本。进行甲基化特异性PCR(MSP)以评估APC基因的启动子甲基化状态。RT-qPCR检测APC的mRNA表达,β-连环蛋白,c-Myc和CyclinD1,同时通过Westernblot分析其蛋白表达。
结果:肝癌患者APC基因甲基化阳性率(46.67%)高于健康供者(11.11%)。与TNMI-II期患者相比,TNMIII-IV期患者的APC基因表现出明显的超甲基化,APC基因甲基化状态与肿瘤大小和淋巴结转移相关,而APC基因甲基化与患者的性别和年龄无关。APC甲基化可能与APC表达水平有关,HCC细胞中的APC表达被异常启动子超甲基化沉默。在甲基化APC的HCC患者中,β-连环蛋白的mRNA和蛋白表达,c-Myc和CyclinD1高于未甲基化患者亚组和健康供体。
结论:肝癌样本中APC的下调与启动子高甲基化相关。APC甲基化可作为肝癌诊断的新生物标志物,与Wnt/β-Catenin信号通路的调控有关。