Effect of cadmium on TET enzymes and DNA methylation changes in human embryonic kidney cell
10.3760/cma.j.issn.0253-9624.2015.09.011
- VernacularTitle:镉暴露对人胚肾细胞TET酶表达及DNA甲基化水平的影响
- Author:
Jinhui LI
1
,
2
;
Wenxue LI
;
Hua YIN
;
Bo ZHANG
;
Wei ZHU
Author Information
1. 510080广州，中山大学公共卫生学院预防医学系
2. 广州市疾病预防控制中心毒理学检验部
- Keywords:
Cadmium;
DNA methylation;
TET protein;
Cell
- From:
Chinese Journal of Preventive Medicine
2015;(9):822-827
- CountryChina
- Language:Chinese
-
Abstract:
Objective To detect the expression changes of the demethylase TETs (Ten-eleven translocation enzymes) in human embryonic kidney cell (HEK293) exposed to high dose cadmium chloride (CdCl2), and to investigate the regulation effects of TETs on global genomic methylation. Methods HEK293 cells were exposed to CdCl2 for 24 h, 48 h and 72 h, the survival rate was tested by CCK-8 (cell counting kit-8) method, and the cell morphology was observed. The levels of TETs mRNA and protein were detected by fluorescence quantitative PCR and Western blot, respectively. The genomic DNA methylation level was detectedby pyro sequencing assay. Results CdCl2 had toxic effects on HEK293 cells, and the half inhibitory concentration (IC50) was 1.78 μmol/L. After exposure of CdCl2 for 24 h, 48 h and 72 h, the morphology of HEK293 cells was altered, and the high dose group (2.0 μmol/L) showed vacuolar changes
and fuzzy appearance. The level of TET1 mRNA in groups of 0.0,0.5,1.0,2.0μmol/L were 0.23±0.13,0.48± 0.12,0.59±0.16 and 0.95±0.39, respectively (F=182.89,P=0.002);The level of TET2 mRNA in groups of 0.0, 0.5,1.0,2.0μmol/L were 0.23±0.12,0.32±0.02,0.31±0.10 and 0.34±0.07, respectively (F=27.94,P<0.001);The level of TET3 mRNA in groups of 0.0,0.5,1.0,2.0 μmol/L were 0.26 ± 0.10,0.27 ± 0.11,0.25 ± 0.11 and 0.28 ± 0.09, respectively (F=1.76,P=0.036).The interaction effect existed between exposure time and doses of TET1 mRNA,TET2 mRNA and TET3 mRNA (F values were 32.94,23.04 and 13.78,respectively;P values were <0.001,0.041 and <0.001,respectively).Western blot showed that in different exposure time and dose, the protein expression levels of TETs had the similar trend as mRNA levels.In 24 h (55.01 ± 3.62)%,48 h (48.31±8.99)%, 72 h (48.76±6.60)%, the DNA methylation had significant differences(F=18.50,P<0.001);In groups of 0.0 μmol/L(55.29±2.83)%,0.5 μmol/L(55.35±3.11)%,1.0 μmol/L(48.58±6.40)% and 2.0 μmol/L(43.56 ± 7.89)%, the differences of DNA methylation had significant differences(F=7.03,P=0.048); the effect of interaction was also existed (F=2.73,P=0.043). Conclusion In the short term exposure to CdCl2, the levels of TETs mRNA and protein showed a trend of increase according to the exposure time and dose, and the methylation level of whole genomic DNA was also altered. The demethylase TETs may play a role in regulating the genomic methylation level of HEK293 exposed to cadmium.
