Influence of smoking on methylation and hydroxymethylation levels in global DNA and specific sites of KRT14, KRT19, MIR-9-3 and MIR-137 genes of oral mucosa.

Objective: The aim of this study was to investigate the influence of smoking on the methylation and hydroxymethylation status of global DNA and specific sites of KRT14, KRT19, miR-9-3 and miR-137 genes in the healthy oral mucosa.

Methods: Samples of oral epithelial cells were collected using mouthwash from a population of former and current smokers and those who had never smoked. Genomic DNA was extracted, and global DNA methylation and hydroxymethylation was performed using an ELISA-based technique; DNA methylation at specific sites was performed using Methylation-Specific PCR (MSP) (KRT14, miR-9-3 and miR-137) or Methylation-Sensitive Restriction Enzymes (MSRE) (KRT19). K14 and K19 protein expression was analysed by immunohistochemistry.

Results: Higher levels of global DNA methylation were found in current smokers with over 15 years of consumption (p=0.04), but no differences were found in relation to global DNA hydroxymethylation. No differences in global DNA methylation and hydroxymethylation levels were found in relation to age or gender. Global DNA methylation was higher than the hydroxymethylation level (p<0.001) but they were not correlated in the oral mucosa. For specific sites, miR-9-3 hypomethylation was detected in current smokers (p<0.001). Additional analysis showed no difference in the methylation status when age, gender, period of consumption or amount of cigarettes were considered for any of the studied genes. K19 expression was higher in current smokers in comparison to former smokers and those who had never smoked (p<0.05).

Conclusion: We concluded that smoking habits were capable of inducing changes in global DNA methylation, miR-9-3 methylation status and K19 expression.