Effect Of SOD2 On Cisplatin Resistance And Invasion Of Human Lung Adenocarcinoma Stem Cells

Objective:1.The expression levels of protein and mRNA of Superoxide dismutase 2(SOD2)in lung adenocarcinoma A549 cells and A549 stem cells were detected.2.To preliminarily investigate the effects of SOD2 on the regulation of lung adenocarcinoma A549 stem cells,cisplatin resistance and invasion and metastasis.Methods:1.A549 cells were cultured in 10%FBS medium,and the tightly packed monoclonal A549 cells were selected by infinite dilution method,and the second generation of A549 stem cell pellets were cultured in SFM medium.2.CCK8 kit was used to detect the proliferation differences between A549 cells and A549 stem cell spheres,the changes in survival rate of the two cells at different cisplatin concentrations,and the proliferation of A549 stem cell spheres after transfection with siRNA-SOD2.3.The kit was used to detect the ROS content and SOD2 concentration of A549 cells and A549 stem cells at different cisplatin concentrations,and the ROS content changes after transfection of A549 stem cells with siRNA-SOD2.4.Western Blot and RT-PCR were used to detect the expression levels of SOD2 protein and mRNA in A549 cells and A549 stem cells,and to detect the differences in the expression levels of various transcription factors(Bmi-1,Nanog,Sox2,Oct4)and apoptotic proteins(Bax,Bcl-2,Caspase3)in the two cells,as well as the changes in the expression levels of SOD2,Bax,Bcl-2,Caspase3 in A549 stem cells under different cisplatin concentrations.5.The A549 stem cell spheres were transfected with siRNA-SOD2,and the expression levels of interfering SOD2 protein and mRNA were detected by Western blot and RT-PCR to verify the transfection efficiency and screen out the most appropriate interfering fragments.6.Western blot was used to detect the protein expression of various transcription factors in A549 stem cells after interference with SOD2 gene,and the change of tumor stem cell sphericity was observed.7.The A549 stem cell spheres were detected by Western blot and flow cytometry(FCM)via siRNA-SOD2.8.Differences in emt-related proteins(E-cadherin,Vimentin)between A549 cells and A549 stem cells were detected by Western blot,and changes in E-cadherin and Vimentin proteins were detected after transfection of siRNA-SOD2 with A549 stem cells.9.Transwell test was used to detect the difference in invasion of A549 cells and A549 stem cells,as well as the change in the number of cell invasion after transfection of siRNA-SOD2 with A549 stem cells.Results:1.The proliferation capacity of A549 cells and A549 stem cells significantly decreased with the increase of cisplatin concentration,and the ROS content and SOD2 concentration gradually increased.Compared with A549 cells,the proliferation,anti-apoptotic ability and SOD2 concentration of A549 stem cells were significantly higher,while the ROS content was lower.2.The expression levels of SOD2,Bax and Caspase3 proteins in A549 stem cells gradually increased along with the cisplatin concentration,while the expression levels of bcl-2 proteins gradually decreased.3.Compared with A549 cells,the expression levels of SOD2 protein and mRNA in A549 stem cell spheres were higher,and the expression levels of bmi-1,Nanog,Sox2 and Oct4 were also higher.4.After transfection with siRNA-SOD2 with A549 stem cell spheres,the expression levels of SOD2 protein and mRNA decreased,the expression levels of dry factor proteins(Bmi-1,Nanog,Sox2,Oct4)decreased,and the proliferation rate and re-sphericity of tumor stem cells decreased,while the ROS content increased.5.Western blot and FCM results showed that the apoptosis rate of cells transfected with siRNA-SOD2 and cisplatin by A549 stem cell pellets was significantly increased.6.The Transwell test and Western Blot results showed that the invasion and metastasis ability of A549 cells was lower than that of A549 stem cells,and the number of invasion ability of A549 stem cells decreased after transfection with siRNA-SOD2.Conclusion:1.The concentration of A549 cells and A549 stem cells in lung adenocarcinoma increased along with cisplatin,the cell proliferation capacity decreased,and the ROS content and SOD2 concentration gradually increased.2.Compared with lung adenocarcinoma A549 cells,the expression levels of SOD2 protein and mRNA in lung adenocarcinoma stem cells were higher.3.SOD2 is one of the reasons for maintaining cisplatin resistance and invasion of human lung adenocarcinoma A549 stem cells.