大鼠肝癌组织中DLC1、ASC、p16和DLK1基因甲基化状态与肝癌的关系

Analysis of DLC1,ASC,p16 and DLK1 gene methylation in a rat model of liver cancer

目的探讨大鼠肝癌组织中DLC1、ASC、p16和DLK1基因启动子区甲基化状态与肝癌发生的关系。方法 55只Wistar雄性大鼠随机分为黄曲霉毒素B1(Aflatoxin B1,AFB1)实验组(35只)和空白对照组(20只),用AFB1诱发大鼠肝癌并建立大鼠实验动物肝癌模型。用甲基化特异性PCR(MS-PCR)技术和琼脂糖凝胶电泳方法检测大鼠肝癌组织及正常肝脏组织中DLC1、ASC、p16和DLK1基因启动子区的甲基化情况,分析4个基因的甲基化状态与肝癌发生的关系。结果在实验第52周可见大鼠肝脏发生典型的肝癌病理学改变,实验诱发大鼠肝癌模型制作成功。MS-PCR技术检测显示在大鼠肝癌组织中DLC1、ASC、p16和DLK1基因启动子区的甲基化率分别为83.3%、93.3%、86.7%和10.0%,在大鼠正常肝脏组织中的甲基化率分别为14.3%、35.7%、21.4%和85.7%,二者比较差异均有统计学意义(P均<0.05)。琼脂糖凝胶电泳法检测显示4种基因甲基化均为阳性。结论大鼠肝癌组织中DLC1、ASC、p16基因启动子区高度甲基化,DLK1基因启动子呈低甲基化水平,提示DLC1、ASC、p16和DLK1基因启动子区的异常甲基化与大鼠肝癌发生的关系密切。

Objective To study the potential association between liver cancer and methylation of the promoters of the genes DLC1, ASC,p16,and DLK1. Methods Primary hepatocellular carcinoma （PHC）was induced in 35 Wistar rats by injection of AFB1. Another group of 20 rats received no drug and served as a negative control. At week 52,all rats were killed and tissue samples were harvested and analyzed for histopathology. Methylation-specific PCR was used to analyze methylation of the promoters of the genes DLC1 ,ASC, p16,and DLK1 in rat liver in both groups. Results AFBl-induction of PHC led to the expected pathological changes in rat liver. Methylation rates of DLC1 ,ASC,p16 in liver tissue were significantly higher in rats with PHC（ 83.3% ,93.3%, 86.7% ,respectively） than in control rats （14.3%,35.7%,21A%;all P〈0.05）,whereas the opposite was true for methylation rates of DLK1 （10.0% vs 85.7%, P〈0.05 ）. Conclusion PHC may be associated with upregnlation of DLC1 ,ASC,and p16 methylation,as well as downregulation of DLK1 methylation. Thus, aberrant methylation of DLC1 ,ASC,p16 and DLK1 may contribute to PHC onset and progression.