Expression and DNA methylation status of the Rap2B gene in human bronchial epithelial cells treated by cigarette smoke condensate

Inhal Toxicol. 2015;27(10):502-9. doi: 10.3109/08958378.2015.1076546. Epub 2015 Aug 26.


Background: The relationship between lung cancer and smoking has been demonstrated. The Rap2B gene is usually overexpressed in lung cancers. This study was aimed to investigate the Rap2B gene expression and its promoter methylation in human bronchial epithelial cells (16HBE) treated by cigarette smoke condensate (CSC).

Methods: 16HBE cells were treated with CSC (1/8 IC50). Soft ager assay, tumorigenicity test, chromosome aberrations analysis were used to identify the transformed cells. The expression level of mRNA and protein of Rap2B was detected using real time PCR and Western blotting, respectively. The genome DNA methylation level was detected using combined bisulfite restriction analysis (COBRA) and the methylation status of the target fragment in Rap2B gene promoter was determined by bisulfite sequencing PCR (BSP).

Results: The 16HBE cells were successfully malignant transformed after the chronic exposure to CSC. The expression of Rap2B gradually increased in the process of malignant transformation. Meanwhile, global DNA was hypomethylated. However, no obvious change was observed in the methylation level of Rap2B gene promoter in transformed 16HBE cells.

Conclusions: Rap2B gene may play an important role in the process of lung cancer and global DNA hypomethylation might be an early event in tumorigenesis.

Keywords: Cigarette smoke condensate; DNA methylation; Rap2B gene; lung cancer.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bronchi / cytology
  • Cell Line
  • Cell Transformation, Neoplastic
  • DNA Methylation / drug effects*
  • Epithelial Cells / drug effects*
  • Epithelial Cells / metabolism
  • Humans
  • Promoter Regions, Genetic
  • Smoke / adverse effects*
  • Tobacco*
  • rap GTP-Binding Proteins / genetics*
  • rap GTP-Binding Proteins / metabolism


  • Smoke
  • RAP2B protein, human
  • rap GTP-Binding Proteins