Wang Shan, He Zhini, Li Daochuan, Zhang Bo, Li Miao, Li Wenxue, Zhu Wei, Xing Xiumei, Zeng Xiaowen, Wang Qing, Dong Guanghui, Xiao Yongmei, Chen Wen, Chen Liping
Department of Toxicology, Guangzhou Key Laboratory of Environmental Pollution and Health Risk Assessment, School of Public Health, Sun Yat-sen University, Guangzhou, China.
Departmant of Toxicology, Guangzhou Center for Disease Control and Prevention, Guangzhou, China.
Toxicology. 2017 Jun 15;385:1-9. doi: 10.1016/j.tox.2017.04.011. Epub 2017 Apr 27.
Chronic exposure to aflatoxin B (AFB) is linked to the development of hepatocellular carcinoma (HCC). To identify differentially methylated genes involved in AFB-induced cell transformation, we analyzed DNA methylation patterns in immortal human hepatocyte L02 cells expressing an oncogenic H-Ras allele (L02R cells) and AFB-transformed L02R (L02RT-AFB) cells by performing genome-wide methylation profiling. We treated L02R cells with 0.3μM AFB weekly and observed a transformed phenotype at the 17th week post-treatment. The transformed cells (L02RT-AFB) could grow in an anchorage independent fashion and form tumors in immunodeficient mice. qRT-PCR was performed to examine whether gene methylation led to a reduction in gene expression of methylated candidate genes. As a result, the expression of the following seven genes including JUNB, RUNX3, NAV1, CXCR4, RARRES1, INTS1, and POLL was down-regulated in transformed L02RT-AFB cells. The reduction of gene expression of these genes could be reversed by treatment of 5-azadeoxycytidine. The methylated CpG sites of RUNX3 genes were verified using bisulfite sequencing PCR (BSP) assay. Furthermore, a dynamic change in RUNX3 methylation was observed over the course of AFB-induced cell transformation, which was corresponded to the alteration of gene expression and the extent of DNA damage. In vitro study showed that methylation of RUNX3 tended to abate in L02R cells treated with AFB for a short-term period of time. Notably, hypermethylation of RUNX3 appeared in 70% (14/20) of human hepatocellular carcinomas. Moreover, LINE-1 hypomethylation and dynamic changes of DNMTs, TETs and MeCP2 expression were also observed during AFB-induced transformation. Taken together, these observations suggest that aberrant methylation of RUNX3 and LINE-1 might be involved in AFB-induced carcinogenesis.
长期接触黄曲霉毒素B（AFB）与肝细胞癌（HCC）的发生有关。为了鉴定参与AFB诱导细胞转化的差异甲基化基因，我们通过进行全基因组甲基化分析，分析了表达致癌性H-Ras等位基因的永生化人肝细胞L02细胞（L02R细胞）和AFB转化的L02R（L02RT-AFB）细胞中的DNA甲基化模式。我们每周用0.3μM AFB处理L02R细胞，并在处理后第17周观察到转化表型。转化细胞（L02RT-AFB）能够以不依赖贴壁的方式生长，并在免疫缺陷小鼠中形成肿瘤。进行qRT-PCR以检查基因甲基化是否导致甲基化候选基因的基因表达降低。结果，包括JUNB、RUNX3、NAV1、CXCR4、RARRES1、INTS1和POLL在内的以下七个基因的表达在转化的L02RT-AFB细胞中下调。这些基因的基因表达降低可通过5-氮杂脱氧胞苷处理逆转。使用亚硫酸氢盐测序PCR（BSP）分析验证了RUNX3基因的甲基化CpG位点。此外，在AFB诱导的细胞转化过程中观察到RUNX3甲基化的动态变化，这与基因表达的改变和DNA损伤程度相对应。体外研究表明，在短期用AFB处理的L02R细胞中，RUNX3的甲基化趋于减弱。值得注意的是，RUNX3的高甲基化出现在70%（14/20）的人类肝细胞癌中。此外，在AFB诱导的转化过程中还观察到LINE-1低甲基化以及DNMTs、TETs和MeCP2表达的动态变化。综上所述，这些观察结果表明RUNX3和LINE-1的异常甲基化可能参与AFB诱导的致癌作用。
