Aberrant methylation of RUNX3 is present in Aflatoxin B1-induced transformation of the L02R cell line

Chronic exposure to aflatoxin B-1 (AFB(1)) is linked to the development of hepatocellular carcinoma (HCC). To identify differentially methylated genes involved in AFB(1)-induced cell transformation, we analyzed DNA methylation patterns in immortal human hepatocyte L02 cells expressing an oncogenic H-Ras allele (L02R cells) and AFB(1)-transformed L02R (L02RT-AFB(1)) cells by performing genome-wide methylation profiling. We treated L02R cells with 0.3 mu M AFB(1) weekly and observed a transformed phenotype at the 17th week post-treatment. The transformed cells (L02RT-AFB(1)) 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(1) 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(1)-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(1) 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(1)-induced transformation. Taken together, these observations suggest that aberrant methylation of RUNX3 and LINE-1 might be involved in AFB(1)-induced carcinogenesis.