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 mCmC, uCuC, mCuC and uCmC. Of these, mC and uC represent methylated and unmethylated CpG, respectively. The DNA bisulphite sequence demonstrated that most CpGs of mCmC and uCuC were methylated and unmethylated, respectively. Nevertheless, some CpGs of each mCuC or uCmC 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 %mCmC and %uCuC increased, while the %mCuC decreased in current smokers (p = 0.002, 0.015, and <0.0001, respectively). Additionally, the lower %mCuC still persisted in persons who had stopped smoking for over 1 year (p = 0.001). The %mCuC also decreased in the higher pack-year smokers (p = 0.028). Smoking possibly altered mCuC to mCmC and uCuC forms, and changes uCmC to uCuC 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.
Highlights
Tobacco smoking is a predisposing factor of many malignancies [1,2,3,4]
We found that the odds ratio (OR) was 6.90 and the 95% confidential interval (CI) was 2.53–18.82 with p,0.0001 (Figure 4A)
This study confirms that COBRALINE-1 does analyse the overall methylation level, but it is able to show the methylation pattern of long interspersed element-1 (LINE-1).We found that uCuC and mCmC represent the hypomethylation and hypermethylation of LINE-1, respectively
Summary
The risk of upper aerodigestive cancers increases with the higher pack-years cigarette smoking [3,5,6]. This risk decreases after discontinuation of smoking and reverts to the nonsmoker risk level if smoking is ceased for more than 15 years [3,6]. Smoking increases the number of keratinised cells in the epithelium of the tongue and hard palate [7]. This effect varied in different regions, depending on the extent of direct exposure to smoke [8]. Oral mucosal lesions resolved after cessation of smoking for a period of time [9,10]
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