G9a在砷致HaCaT细胞DNA损伤中的作用初探
Preliminary study on role of G9a in DNA damage in HaCaT cells induced by arsenic
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摘要:
背景 砷可导致皮肤癌、肺癌等，相关机制存在多种假说。有研究认为DNA损伤及其修复抑制在砷的毒作用机制中起重要作用。本课题组前期在人群研究中也发现砷暴露可引起外周血淋巴细胞DNA双键断裂损伤。因此，探讨砷暴露引起细胞DNA损伤的分子机制对研究砷致癌机制尤为重要。
目的 通过检测人永生化皮肤角质形成细胞（HaCaT细胞）组蛋白赖氨酸甲基转移酶2（G9a）过表达前后NaAsO2所致细胞DNA损伤，初步探讨G9a与砷致HaCaT细胞DNA损伤修复关系。
方法 体外常规培养HaCaT细胞，以0.00、2.50、5.00、10.00 μmol/L NaAsO2处理HaCaT细胞24 h，其中0.00 μmol/L为空白对照组。采用实时荧光定量PCR法检测G9a mRNA转录水平，免疫印迹法检测G9a蛋白表达水平，单细胞凝胶电泳法检测各组HaCaT细胞DNA损伤水平。为了进一步验证G9a对NaAsO2致细胞DNA损伤的影响，用pCDNA3.1-G9a质粒瞬时转染HaCaT细胞的基础上，再以0.00 μmol/L或10.00 μmol/L NaAsO2处理细胞24 h，另设立空白对照组、空载体转染组和单独染砷组（10.00μmol/L），采用上述实验方法检测各项指标的变化。
结果 用不同浓度NaAsO2染毒HaCaT细胞24 h后，实时荧光定量PCR结果显示，与空白对照组（1.00±0.00）比较，2.50、5.00、10.00 μmol/L染砷组G9a mRNA转录水平（0.87±0.04、0.70±0.05、0.59±0.04）逐渐减低（P < 0.05）；免疫印迹法结果显示，G9a蛋白表达水平在5.00、10.00 μmol/L染砷组（0.40±0.05、0.29±0.06）较对照组（0.59±0.09）降低；单细胞凝胶电泳结果显示，细胞彗星尾部DNA百分含量（0.63±0.07、3.26±0.34、6.79±0.66、13.18±2.34）和Olive尾矩（1.08±0.05、3.67±0.20、6.26±0.46、12.63±0.63）均随NaAsO2浓度增加而逐渐增加（r=0.93、0.95，P < 0.05）。质粒转染HaCaT细胞后，实时荧光定量PCR和Western blot结果显示，与对照组（1.00±0.00、0.67±0.06）比较，G9a高表达组G9a mRNA的转录水平（105.93±28.72）及蛋白表达水平（1.07±0.06）均明显增加（P < 0.05），与染砷组（0.57±0.08、0.40±0.02）比较，G9a高表达联合染砷组G9a mRNA的转录水平（41.80±7.14）及蛋白表达水平（0.67±0.02）也有所升高（P < 0.05）；单细胞凝胶电泳结果显示，与空白对照组（0.91±0.10、0.53±0.09）比较，G9a高表达组细胞的彗星尾部DNA百分含量（1.00±0.18）及Olive尾矩（0.77±0.13）无明显变化，与染砷组（11.41±1.02、12.59±1.38）比较，G9a高表达联合染砷组细胞彗星尾部DNA百分含量及Olive尾矩（15.24±1.25、17.12±2.88）有所升高（P < 0.05）。
结论 砷可导致HaCaT细胞DNA损伤和G9a mRNA转录水平及蛋白表达水平的改变，但还不确定G9a的变化是否与砷所致DNA损伤机制有关联，其机制有待进一步研究。
Abstract: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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