LINE-1 Hypomethylation in a Choline-Deficiency-InducedLiver Cancer in Rats: Dependence on Feeding Period

[摘要] Chronic feeding of methyl-donor (methionine, choline, folic acid,and vitamin B12) deficient diet induces hepatocellular carcinomaformation in rats. Previous studies have shown that promoter CpGislands in various cancer-related genes are aberrantly methylatedin this model. Moreover, the global genome inmethyl-donor-deficient diet fed rats contains a lesser amount of5-methylcytosine than control livers. It is speculated that morethan 90% of all 5-methylcytosines lie within the CpG islands ofthe transposons, including the long/short interspersed nucleotideelements (LINE and SINE). It is considered that the5-methylcytosines in LINE-1 limit the ability of retrotransposonsto be activated and transcribed; therefore, the extent ofhypomethylation of LINE-1 could be a surrogate marker for aberrantmethylation in other tumor-related genes as well as genomeinstability. Additionally, LINE-1 methylation status has beenshown to be a good indicator of genome-wide methylation. In thisstudy, we determined cytosine methylation status in the LINE-1repetitive sequences of rats fed a choline-deficient (CD) diet forvarious durations and compared these with rats fed acholine-sufficient (CS) diet. The methylation status of LINE-1 wasassessed by the combined bisulfite restriction analysis (COBRA)method, where the amount of bisulfite-modified and RsaI-cleavedDNA was quantified using gel electrophoresis. Progressivehypomethylation was observed in LINE-1 of CD livers as a functionof feeding time; that is, the amount of cytosine in total cytosine(methylated and unmethylated) increased from 11.1% (1 week) to19.3% (56 weeks), whereas in the control CS livers, it increasedfrom 9.2% to 12.9%. Hypomethylation in tumor tissues wasslightly higher (6%) than the nontumorous surrounding tissue. Thepresent result also indicates that age is a factor influencing theextent of cytosine methylation.

[学科分类] 基础医学