Objective To observe the influences of different doses of sodium arsenite on histone acetylation regulation, mRNA transcription and protein expression of O6-methylguanine-DNA methyltransferase gene(MGMT)in HaCaT cells.
Methods HaCaT cells were treated with 25.00, 12.50, 6.25 and 3.13 μmol/L NaAsO2 for 72 h at intervals and repeatedly. Histone acetylation modifications in two transcription regulatory region(ChIP1, ChIP2 region)and in coding region(ChIP3 region, the control region)of MGMT gene were detected by chromatin immuno-precipitation combined with quantitative PCR, the mRNA transcription and the protein expression of MGMT were detected by real-time quantitative PCR and Western blot. HaCaT cells untreated with NaAsO2(0.00 μmol/L)were set as the blank control group, human epidermal squamous carcinoma cell line A431 cells were set as the positive control group.
Results Among the groups of HaCaT cells treated with 0.00, 3.13, 6.25, 12.50 and 25.00 μmol/L NaAsO2, the levels of histone acetylation of H3K9 in ChIP1 transcription regulatory region of MGMT gene were 176.68& #177;8.50, 175.71& #177;18.14, 161.26& #177;16.28, 146.23& #177;24.00 and 82.64& #177;33.87 respectively, the differences were significant(F=28.809, P<0.05). The levels of histone acetylation of H4 in ChIP1 transcription regulatory region were 183.59& #177;11.98, 180.84& #177;24.10, 166.52& #177;5.48, 156.87& #177;10.64 and 103.42& #177;7.04, the differences were significant(F=36.493, P<0.05). The levels of histone acetylation of H3K9 in ChIP2 transcription regulatory region were 171.11& #177;16.54, 167.55& #177;8.97, 156.51& #177;8.59, 135.88& #177;16.55 and 82.01& #177;3.96, the differences were significant(F=49.626, P<0.05). The levels of histone acetylation of H4 in ChIP2 transcription regulatory region were 117.23& #177;16.21, 143.29& #177;10.59, 135.87& #177; 7.44, 105.48& #177;7.56 and 78.79& #177;6.92, the differences were significant(F=25.438, P<0.05). The levels of histone acetylation of H3K9 in ChIP3 coding region were 37.53& #177;6.23, 35.57& #177;5.85, 40.81& #177;4.45, 42.18& #177;1.23 and 41.87& #177;5.71, the differences were not significant(F=2.341, P>0.05). The levels of histone acetylation of H4 in ChIP3 coding region were 40.78& #177;2.42, 38.56& #177;4.66, 39.47& #177;2.88, 33.13& #177;3.48 and 31.48& #177;4.99, the differences were not significant(F=3.027, P>0.05).The levels of MGMT mRNA transcription were 1.198 29& #177;0.159 97, 1.518 31& #177;0.180 54, 1.425 22& #177;0.180 39, 1.014 54& #177;0.096 79 and 0.887 72& #177;0.020 00, the differences were significant(F=37.359, P<0.05). The levels of MGMT protein expression were 1.066 19& #177;0.061 24, 1.174 47& #177; 0.064 75, 0.848 83& #177;0.057 01, 0.471 63& #177;0.023 34 and 0.240 34& #177;0.014 43, the differences were significant(F=20.687, P<0.05).
Conclusion Arsenic can cause histone deacetylation of transcription regulatory region of MGMT gene, which results in MGMT mRNA transcription and protein expression to be inhibited. It might be one of important mechanisms of arsenic-induced skin lesion.
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