The Role Of Transcription Factor Nrf2 On The Cell Apoptosis In Arsenic-induced Malignant Transformation Of Cells

ObjectiveTo explore the mechanism of human bronchial epithelial cell?HBE cells?and human keratinocytes?HaCaT cells?malignantly transformed by long term exposure of sodium arsenite?NaAs O₂,1.0?M?,and the effects on the cell apoptosis rate and protein expression apoptosis related to this process.And research the role of Nrf2 in this process,for the further study of arsenic carcinogenic mechanism provides the necessary basic information.Methods1.The cell model:Use the early stage of laboratory established HBE,HaCaT cells malignant transformation model as the research object.With 0.0 and 1.0?mol/L NaAsO₂ DMEM sugar medium culture HBE,HaCaT cells to 43 and 35 generation,respectively,At this point,the cell malignant transformation have occurred.In treatment HBE cells of 0,1,8,15,22,29,36,43 generations,HaCaT cells of 0,1,7,14,21,28,35 generations as the dynamic monitoring spot,respectively.2.Detection of the change in the cell apoptosis rate and apoptosis related proteins in the process of NaAsO₂ cause cell malignant transformation:Using flow cytometry instrument to detect the changes of cell apoptosis rate in each monitoring spot in the process of NaAsO₂ cause HBE,HaCaT cells malignant transformation;Using Western blot method to detect apoptosis raleted protein Caspase-3,Cleaved-caspase-3,Caspase-8,Cleaved-caspase-8,Caspase-12,Cleaved-caspase-12,CHOP,Bcl-2,Bax,Mcl-1 protein expression level in each monitoring spot in the process of NaAsO₂ cause HBE,HaCaT cells malignant transformation.3.Detection of the effects of transcription factor Nrf2 on cell apoptosis in the process of NaAsO₂ cause cell malignant transformation:Using treatment HBE cells 43generation and HaCaT cells 35 generation for soft AGAR cloning experiment,respectively.pick up a single clone HBE,HaCaT cells in the soft AGAR cloning experiment positive cells?30 or higher?,continue to expand culturing.respectively named malignant transformation of HBE Cells?T-HBE Cells?and malignant transformation of HaCaT Cells?T-HaCaT Cells?.Again,respectively,transfection Nrf2siRNA to T-HBE and T-HaCaT cells,make the Nrf2 gene express silence.at the same time set up corresponding to the extend control group,the malignant transformation group,and the transfection Con siRNA group,respectively,as the negative control group,positive control group and blank control group.After using Western blot tests the result of Nrf2 gene express silence in T-HBE,T-HaCaT cells,using flow cytometry instrument detect the change of cell apoptosis rate;Using enzyme standard instrument detect the level of hydrogen peroxide?H₂O₂?expression;Meanwhile using Western blot detect the expression of apoptosis related protein Cleaved-caspase-3,Caspase-3,Cleaved-caspase-8,Caspase-8,Cleaved-caspase-12,Caspase-12,CHOP,Bcl-2,Bax and Mcl-1 protein.Results1.The change of cell apoptosis rate and apoptosis rated protein in the process of NaAsO₂ cause cell malignant transformation:?1?Cultured each monitoring spot of HBE,HaCaT cells were detected the change of apoptosis rate,results showed that compared with control group 0 generation cells,the apoptosis rate is obvious downward trend,respectively after infected 22 generations in HBE cells and after infected 7 generations in HaCaT cells,the differences were statistically significant?P<0.05??2?Cultured each monitoring spot of HBE,HaCaT cells were detected the change of apoptosis rate,results showed Caspase-3?Cleaved-caspase-3 protein and Cleaved-caspase-3/Caspase-3 showed downward trend in HBE,HaCaT cells.In HBE cells,compared with control group 0 generation cells and the extend cells control group,Caspase-3,Cleaved-caspase-3 protein are all after infected 22 generations,Cleaved-caspase-3/Caspase-3 are respectively after infected 15 generations and after infected 29 generations,appear the low expression state?P<0.05?.In HaCaT cells,compared with control group 0 generation cells and the extend cells control group,Caspase-3 protein and Cleaved-caspase-3/Caspase-3 are all after infected 21generations,Cleaved-caspase-3 protein are respectively after infected 14 generations and after infected 21 generations,appear the low expression state?P<0.05?.?3?Cultured each monitoring spot of HBE,HaCaT cells were detected the change of apoptosis related proteins in different apoptotic pathways in the process of NaAsO₂cause cell malignant transformation,results showed that Cleaved-caspase-8?Caspase-8?Cleaved-caspase-12?Caspase-12 protein do not have change in this process.In HBE cells,compared with control group 0 generation cells and the extend cells control group,CHOP proteins are all at after infected 15 generations,and Bax proteins are all after infected 29 generations,have a significant reduction?P<0.05?;The Bcl-2,Mcl-1proteins are all after infected 15 generation show clear rising trend?P<0.05?.In HaCaT cells,compared with control group 0 generation cells and the extend cells control group,CHOP,Bax protein are all after infected 21 generations,have a state of low expression?P<0.05?;The Bcl-2 protein are respectively after infected 7 generation and after infected 14 generation,Mcl-1 protein are all after infected 14 generations,have a state of high expression?P<0.05?.2.Detection of the effects of transcription factor Nrf2 on cell apoptosis in the process of NaAsO₂ cause cell malignant transformation:?1?Detection of result of Nrf2 gene silence after transfection Nrf2 si RNA to T-HBE and T-HaCaT cells,results show that compared with control group of extend HBE and HaCaT cells,in T-HBE and T-HaCaT cells,the expression of Nrf2 proteins are significantly increased?P<0.05?;Compared with control group of transfection Con siRNA,the expression of Nrf2 proteins are significantly decreased in the group of transfection Nrf2 siRNA to T-HBE and T-HaCaT cells?P<0.05?.?2?Detection of cell apoptosis rate and H₂O₂ level after transfection Nrf2 siRNA to T-HBE and T-HaCaT cells,results show compared with control group of extend HBE and HaCaT cells,in T-HBE and T-HaCaT cells,the cell apoptosis rate and H₂O₂ level are all significantly decrease?P<0.05?;Compared with control group of transfection Con siRNA,the cell apoptosis rate and H₂O₂ level are all significantly increased in the group of transfection Nrf2 siRNA to T-HBE and T-HaCaT cells?P<0.05?.?3?Detection of apoptosis proteins in each apoptosis pathway after transfection Nrf2 siRNA to T-HBE and T-HaCaT cells,results show compared with control group of extend HBE and HaCaT cells,in T-HBE and T-HaCaT cells,Cleaved-caspase-3,caspase-3,CHOP,Bax protein expression are all significantly decreased,the Bcl-2,Mcl-1 protein expression are all significantly increased?P<0.05?,but Cleaved-caspase-8,Caspase-8,Cleaved-caspase-12,Caspase-12 protein expression are not have obvious change.And compared with control group of transfection Con siRNA,in group of transfection Nrf2 siRNA to T-HBE and T-HaCaT cells,the Cleaved-caspase-3,Caspase-3,CHOP,Bax protein expression are all significantly increased,the Bcl-2,Mcl-1 protein expression are all significantly decreased?P<0.05?,and Cleaved-caspase-8,Caspase-8,Cleaved-caspase-12,Caspase-12 protein expression are not have obvious change.Conclusions1.In the process of 1.0?mol/L NaAsO₂ disposed HBE,HaCaT cells make it occur malignant transformation,the cell apoptosis rate are significantly decreased.2.NaAsO₂ may be through activate the endoplasmic reticulum and mitochondrial apoptosis pathway to control HBE,HaCaT cells apoptosis,leading to malignant transformation.3.Nrf2 may be as the upstream gene involved in NaAsO₂ inhibit HBE,HaCaT cells apoptosis.