Patterns and possible roles of LINE-1 methylation changes in smoke-exposed epithelia

PLoS One. 2012;7(9):e45292. doi: 10.1371/journal.pone.0045292. Epub 2012 Sep 18.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Alleles
  • Base Sequence
  • Cell Transformation, Neoplastic / drug effects
  • Cell Transformation, Neoplastic / genetics
  • CpG Islands*
  • DNA Methylation / drug effects*
  • Epithelium / drug effects*
  • Epithelium / metabolism
  • Epithelium / pathology
  • Female
  • Genomic Instability
  • Humans
  • Long Interspersed Nucleotide Elements*
  • Male
  • Middle Aged
  • Molecular Sequence Data
  • Mouth / drug effects
  • Mouth / metabolism
  • Mouth / pathology
  • Mouth Mucosa / drug effects*
  • Mouth Mucosa / metabolism
  • Mouth Mucosa / pathology
  • Sequence Analysis, DNA
  • Smoking / adverse effects*

Grants and funding

This study was financially supported by the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund). KS is supported by the Grant for Development of New Faculty Staff (Ratchadapiseksomphot Endowment Fund) and the Thailand Research Fund (Young Scientific Researcher Grant No. MRG5380176). Apiwat Mutirangura is supported in part by the Research Chair Grant 2011, the National Science and Technology Development Agency, Thailand, the Four Seasons Hotel Bangkok's 4th Cancer Care charity fun run in coordination with the Thai Red Cross Society and Chulalongkorn University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.