| Objective:To explore the role of ubiquitin protein ligase RING2 in the changes of BPDE-DNA adducts and ATR-CHK1 pathway in human lung derived cells(16HBE,BEAS-2B,VA13)exposed to benzo[a]pyrene.Methods:The expressions of RING2 were inhibited by small interfering RNA(SiRNA)in human lung derived cells to construction of RING2 low expression cell model.The human lung derived cells were exposed to B(a)P at concentrations of 0,1,2,4,8,16,32 μmol/L for 24 hours;and the human lung derived cells were exposed to 16 μmol/L BaP for 0,1,2,4,8,12 and 24 hours.BPDE-DNA adducts were evaluated by fluorescent immunohistochemistry method.The levels of ATR(Ataxia telangiectasi and Rad3 related),CHK1(Checkpoint kinase 1),CHK1 Ser345-p,CDC25A(cell division cycle 25 homolog A),CDC25 A S76-P、CDK2(Cyclin dependent kinase 2)and CDK2 Y15 were detected by Western blot.Results:Fluorescent immunohistochemistry results show: Compared with the wild type control group of three lung-derived cells,the levels of fluorescence intensity were significantly elevated in the three types(WT、Si-NC、Si-RING2)of three lung-derived cells exposed to benzo(a)pyrene(P<0.01),and the fluorescence intensity of three lung-derived cells(SiRNA-RING2)were increased by(41.3%,31.7 and 51.4% respectively)compared with three lung-derived cells(WT)exposed to 16 μmol/L benzo[a]pyrene for 24h(P<0.01).Western-blot results show:Compared with normal control group,the relative expression levels of ATR,CHK1,CHK1 S345-p,CDC25 A S76-p and CDK2 Y15-p in three lung-derived cells(WT)were significantly increased along with increasing concentration and exposure time,while the relative expression levels of CDC25 A and CDK2 were significantly decreased(P<0.01);Compared with normal control group,the relative expression levels of ATR,CHK1,CHK1 S345-p,CDC25 A S76-p and CDK2 Y15-p in three lung-derived cells(Si-RING2)were significantly decreased along with increasing concentration and exposure time,while the relative expression levels of CDC25 A and CDK2 were significantly increased(P<0.01).As analysis of covariance to control exposure concentration factors show,the estimate means of the levels of ATR、CHK1、CHK1 S345-p、CDC25A S76-p and CDK2 Y15-p(0.804、0.823、0.756、0.728 and 0.834 respectively)in 16HBE(siRNA-RING2)cell group was significantly lower than those of 16HBE(WT)cell group(1.473、1.311、1.375、1.387 and 1.331 respectively),the estimate means of the levels of CDC25 A and CDK2(1.324 and 1.293 respectively)in 16HBE(siRNA-RING2)cell group was significantly higher than those of 16HBE(WT)cell group(0.803 and 0.767 respectively)(P<0.01);the estimate means of the levels of ATR、CHK1、CHK1 S345-p、CDC25A S76-p and CDK2 Y15-p(0.775、0.831、0.726、0.800 and 0.677 respectively)in BEAS-2B(siRNA-RING2)cell group was significantly lower than those of BEAS-2B(WT)cell group(1.356、1.334、1.429、1.366 and 1.270 respectively),the estimate means of the levels of CDC25 A and CDK2(1.369 and 1.395 respectively)in BEAS-2B(siRNA-RING2)cell group was significantly higher than those of BEAS-2B(WT)cell group(0.775 and 0.811 respectively)(P<0.01);the estimate means of the levels of ATR、CHK1、CHK1 S345-p、CDC25A S76-p and CDK2 Y15-p(0.831、0.863、0.837、0.666 and 0.838 respectively)in VA13(siRNA-RING2)cell group was significantly lower than those of VA13(WT)cell group(1.322、1.343、1.392、1.450 and 1.506 respectively),the estimate means of the levels of CDC25 A and CDK2(1.325 and 1.324 respectively)in VA13(siRNA-RING2)cell group was significantly higher than those of VA13(WT)cell group(0.830 and 0.793 respectively)(P<0.01).As analysis of covariance to control exposure time factors show,the estimate means of the levels of ATR、CHK1、CHK1 S345-p、CDC25A S76-p and CDK2 Y15-p(0.836、0.751、0.726、0.808 and 0.796 respectively)in 16HBE(siRNA-RING2)cell group was significantly lower than those of 16HBE(WT)cell group(1.264、1.309、1.324、1.389 and 1.430 respectively),the estimate means of the levels of CDC25 A and CDK2(1.255 and 1.396 respectively)in 16HBE(siRNA-RING2)cell group was significantly higher than those of 16HBE(WT)cell group(0.791 and 0.781 respectively)(P<0.01);the estimate means of the levels of ATR、CHK1、CHK1 S345-p、CDC25A S76-p and CDK2 Y15-p(0.715、0.736、0.782、0.800 and 0.807 respectively)in BEAS-2B(siRNA-RING2)cell group was significantly lower than those of BEAS-2B(WT)cell group(1.316、1.342、1.536、1.373 and 1.302 respectively),the estimate means of the levels of CDC25 A and CDK2(1.323 and 1.233 respectively)in BEAS-2B(siRNA-RING2)cell group was significantly higher than those of BEAS-2B(WT)cell group(0.776 and 0.743 respectively)(P<0.01);the estimate means of the levels of ATR、CHK1、CHK1 S345-p、CDC25A S76-p and CDK2 Y15-p(0.744、0.817、0.784、0.743 and 0.820 respectively)in VA13(siRNA-RING2)cell group was significantly lower than those of VA13(WT)cell group(1.399、1.447、1.351、1.369 and 1.291 respectively),the estimate means of the levels of CDC25 A and CDK2(1.369 and 1.315 respectively)in VA13(siRNA-RING2)cell group was significantly higher than those of VA13(WT)cell group(0.819 and 0.835 respectively)(P<0.01).Conclusions:B(a)P leads to an increase in intracellular BPED-DNA adducts,the DNA of SiRNA-RING2 cells are much more sensitivity in B(a)P exposure than that of wild type cell group,It may be indicate that RING2 was involved in DNA repair;B(a)P leads to the activation of the DNA damage response pathway ATR-CHK1-CDC25A-CDK2,which is inhibited in three lung-derived cells(SiRNA-RING2)exposed B(a)P.The above conclusions suggest that ubiquitin protein ligase RING2 was involved in DNA repair by affecting the activity of the DNA damage response pathway ATR-CHK1-CDC25A-CDK2. |
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