Nicotine attenuates global genomic DNA methylation by influencing DNMTs gene expression in human endometrial stromal cells

Abstract

Background: There is increasing evidence indicating an incidence of infertility and also the risk of endometrial cancers among smokers. However, the mechanism underlying nicotine adverse effect on female reproduction remains unclear. Growing evidence has suggested that environmental exposures such as nicotine could modulate the epigenome. No study has yet been published to evaluate the direct effect of nicotine on the epigenome profiling of human endometrial stromal cells (HESC). Herein, we decided to examine the direct effects of nicotine on global genomic DNA methylation status and DNA methyl- transferases (DNMTs) gene expression in HESC. HESC were treated with different doses of nicotine (0 or control, 10^{- 11}, 10^{- 8} and 10^{- 6}) M for 24 h and their genomic global DNA methylation and gene expression of DNMTs (DNMT1, DNMT3A, and DNMT3B) were investigated using ELISA and real-time PCR, respectively.

Results: Nicotine treatments reduced the average level of DNMTs gene expression by 90, 79, and 73.4% in 10^{- 11}, 10^{- 8} and 10^{- 6} M of nicotine treated cells as compared to control cells, respectively (p < 0.05). Also, 10^{- 8} and 10^{- 6} M of nicotine concentrations effectively reduced the amounts of 5-methylated cytosine (5-mC) by 1.09 and 1.87% compared to control cells, respectively (p < 0.05). The 5-mC percentages were positively correlated with the relative cellular DNMTs expression in HESC as verified by the Pearson correlation test.

Conclusion: An interesting possibility raised by the current study is that the reduced genomic global DNA methylation level in HESC may be partly due to the suppression of DNMTs gene expression caused by nicotine in these cells.

Keywords: DNMTs gene expression; Endometrial cancer; Global DNA methylation; Nicotine.

Fig. 1

Phase-contrast pictures of Immunocytochemistry assay: strong staining was seen with primary antibody to vimentin as a marker for stromal cells (200× magnification)

Fig. 2

A. Comparison of the relative expression of DNMT1, DNMT3A and DNMT3B in human endometrial stromal cells that were treated with 10^− 11, 10^− 8 and 10^− 6 M of nicotine, measured by quantitative Real-time-PCR. Expression of each gene was normalized to β-Actin mRNA. The culture-media treated control cells were used as a reference, whose expression levels were set to 1.0, and expressions in all other cells were expressed as an n-fold difference relative to controls. Mean values ± SD of three experiments are given. Bars marked with different letters are significantly different as verified by Tukey’s honestly significant difference multiple comparison test (p < 0.05). B. A summary of the change in the average expression of all three DNMTs (T1/3A/3B) in HESC treated with 10^− 11, 10^− 8 and 10^− 6 M of nicotine (p < 0.05)

Fig. 3

Effects of nicotine at the concentrations of 10^− 11, 10^− 8 and 10^− 6 M on global DNA methylation in HESC. Values represent mean ± SD of three experiments. The Bars marked with different letters represents significantly different from other samples as verified by Tukey’s honestly significant difference multiple comparison test (p < 0.05)

Fig. 4

Correlation of average expression levels of DNMT1, DNMT3A and DNMT3B with global DNA methylation in 10^− 11, 10^− 8 and 10^− 6 M concentrations of nicotine treated human endometrial stromal cells. Correlation between mean expressions of DNMT1/3A/3B and %5-mC in genomic DNA of HESC was verified by Pearson correlation test (r² = 0.1754, p = 0.0417)