Background Arsenic can cause cancers such as skin cancer and lung cancer, and there are many mechanical assumptions. Studies have shown that DNA damage and its repair inhibition play an important role in the mechanism of arsenic toxicity. Our previous population-based studies have also found that arsenic exposure can cause DNA double strand breaks in peripheral blood lymphocytes. Therefore, understanding the molecular mechanism of arsenic exposure-induced DNA damage in cells is a key step in studying the carcinogenic mechanism of arsenic.
Objective This study observes the DNA damage induced by NaAsO2 exposure in immortalized non-tumorigenic human epidermal cells (HaCaT) with over-expressed euchromatic histone-lysine N-methyltransferase 2 (G9a) or not, and to preliminarily explore the relationship between G9a and arsenic-induced DNA damage in HaCaT cells.
Methods HaCaT cells were routinely cultured in vitro and treated with different concentrations of NaAsO2 (0.00, 2.50, 5.00 and 10.00 μmol/L) for 24 h. The 0.00 μmol/L was used as blank control. The mRNA transcription and protein expression of G9a were detected by realtime quantitative PCR and Western blot, respectively. DNA damage in HaCaT cells with different treatments was detected by single cell gel electrophoresis. In order to further verify the effect of G9a on DNA damage induced by NaAsO2, HaCaT cells were transfected with pCDNA3.1-G9a plasmid, and then treated with 0.00μmol/L or 10.00μmol/L NaAsO2 for 24h; related blank control group, null-vector transfection group, and arsenic group (10.00μmol/L) were also set up. The changes of related indicators were detected as mentioned earlier.
Results After exposure to NaAsO2 for 24 h, the real-time quantitative PCR results showed that the mRNA transcription levels of G9a in HaCaT cells of the 2.50, 5.00, and 10.00 μmol/L groups (0.87±0.04, 0.70±0.05, 0.59±0.04) were gradually lower than the level of the blank control group (1.00±0.00) (P < 0.05); the Western blot results showed that the protein expression levels of G9a of the 5.00 and 10.00 μmol/L groups (0.40±0.05, 0.29±0.06) were lower than that of the blank control group (0.59±0.09) (P < 0.05); the single cell gel electrophoresis results showed that the tail DNA% (0.63±0.07, 3.26±0.34, 6.79±0.66, 13.18±2.34) and the Olive tail moment (1.08±0.05, 3.67±0.20, 6.26±0.46, 12.63±0.63) were increased (r=0.93 and 0.95, P < 0.05) with higher NaAsO2 exposure dose. After plasmid transfection into HaCaT cells, the real-time quantitative PCR and Western blot results showed that the mRNA transcription level and the protein expression level of the G9a over-expression group (105.93±28.72, 1.07±0.06) were higher than those of the blank control group (1.00±0.00, 0.67±0.06) (P < 0.05), the levels of the G9a over-expression and arsenic exposure group (41.80±7.14, 0.67±0.02) were also higher than those of the arsenic exposure group (0.57±0.08, 0.40±0.02) (P < 0.05); the single cell gel electrophoresis results showed that the tail DNA% and the Olive tail moment of the G9a over-expression group (1.00±0.18, 0.77±0.13) were not significantly changed compared with the blank control group (0.91±0.10, 0.53±0.09), but the levels of the G9a over-expression and arsenic exposure group (15.24±1.25, 17.12±2.88) were increased compared with the arsenic exposure group (11.41±1.02, 12.59±1.38) (P < 0.05).
Conclusion Arsenic can induce DNA damage and changes in the G9a mRNA transcription and protein expression in HaCaT cells; however, it is not certain yet whether the change of G9a is related to the mechanism of arsenic-induced DNA damage, which needs to be further studied.
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