Patterns and Possible Roles of LINE-1 Methylation Changes in Smoke-Exposed Epithelia

Abstract

Tobacco smoking and reduced methylation of long interspersed element-1 (LINE-1) are crucial in oral carcinogenesis. 5′UTR of human LINE-1 sequence contains several CpG dinucleotides which are methylated in various proportions (0-100%). Methylation levels of many LINE-1s in cancer were reduced, hypomethylated. The hypomethylation of each LINE-1 locus can promote instability of genome and repress expression of a gene located on that same chromosome. This study investigated if cigarette smoking influences LINE-1 methylation of oral mucosal cells. The methylation of human LINE-1 in clinically normal oral mucosa of current smokers was compared to non-smokers. By using the combined bisulphite restriction analysis, each LINE-1 sequence was categorised into 4 patterns depending on the methylation status and location of the two 18-bp successive CpG from 5′ to 3′ including m C m C, u C u C, m C u C and u C m C. Of these, m C and u C represent methylated and unmethylated CpG, respectively. The DNA bisulphite sequence demonstrated that most CpGs of m C m C and u C u C were methylated and unmethylated, respectively. Nevertheless, some CpGs of each m C u C or u C m C allele were methylated. Imaging of the digestion products was used to generate %methylation value. No significant difference in the overall LINE-1 methylation level but the differences in percentages of some methylation patterns were discovered. The % m C m C and % u C u C increased, while the % m C u C decreased in current smokers (p = 0.002, 0.015, and < 0.0001, respectively). Additionally, the lower % m C u C still persisted in persons who had stopped smoking for over 1 year (p = 0.001). The % m C u C also decreased in the higher pack-year smokers (p = 0.028). Smoking possibly altered m C u C to m C m C and u C u C forms, and changes u C m C to u C u C forms. In conclusion, smoking changes methylation levels of partial methylated LINE-1s and increased the number of hypo- and hypermethylated loci. These hypomethylated LINE-1s may possess carcinogenesis potential. Moreover, LINE-1 methylation patterns may be useful for monitoring oral carcinogenesis in smokers. © 2012 Wangsri et al.