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

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 (CC)-C-m-C-m, (CC)-C-u-C-u, (CC)-C-m-C-u and (CC)-C-u-C-m. Of these, C-m and C-u represent methylated and unmethylated CpG, respectively. The DNA bisulphite sequence demonstrated that most CpGs of (CC)-C-m-C-m and (CC)-C-u-C-u were methylated and unmethylated, respectively. Nevertheless, some CpGs of each (CC)-C-m-C-u or (CC)-C-u-C-m 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 % (CC)-C-m-C-m and % (CC)-C-u-C-u increased, while the % (CC)-C-m-C-u decreased in current smokers (p = 0.002, 0.015, and <0.0001, respectively). Additionally, the lower % (CC)-C-m-C-u still persisted in persons who had stopped smoking for over 1 year (p = 0.001). The % (CC)-C-m-C-u also decreased in the higher pack-year smokers (p = 0.028). Smoking possibly altered (CC)-C-m-C-u to (CC)-C-m-C-m and (CC)-C-u-C-u forms, and changes (CC)-C-u-C-m to (CC)-C-u-C-u 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.