The impact of cigarette smoke on activity of single nucleotide polymorphisms of the vascular endothelial growth factor-promoter gene in cells of the upper aerodigestive tract.

Abstract

The vascular endothelial growth factor (VEGF) is involved in tumorigenesis of the upper aerodigestive tract. Different single nucleotide polymorphisms (SNPs) turn the regulation of the VEGF gene into a highly complex process, particularly influenced by exogenic factors like cigarette smoke (CSE). Analysis of the SNP- and CSE-dependent VEGF-gene regulation can help to improve antiangiogenic therapies and prognosis. Therefore, the aim of this study was to analyse the influence of CSE on the SNP-dependent regulation of the VEGF-gene in vitro. Human alveolar epithelial-like type-II cells (A549) were transfected with different SNPs and incubated with CSE. SNP- and CSE-dependent VEGF-promoter activity and mRNA stability was measured using qRT-PCR and Western blot analysis. Transfection with SNP -460 (ATC) and incubation with 10% CSE resulted in +19% elevated VEGF-promoter activity (P<0.05). Transfection with SNP -2578/-460 (CTC) and 10% CSE incubation resulted in a 14% reduction of VEGF-promoter activity (P<0.05). Regarding mRNA stability, transfection with SNP +936 T allele led to half-life of 1.11hours, which decreased to 0.2hours after incubation with CSE. In contrast, on protein level SNP +936 T transfection showed a not significant increase up to 176% (P>0.05), while incubation with CSE led to a significant decrease to 61% (P=0.002). Transcriptional regulation of the VEGF gene by SNP -460 (ATC) in combination with CSE represents a mechanism for elevated VEGF expression and may be associated with a worse prognosis. The influence of +SNP 936 on mRNA stability may be responsible for regulation of VEGF plasma levels.