Study On The Killing Effect Of Endogenous ROS Homeostasis Imbalance On Melanoma Cells

Objection: The expression of CBP and Nrf2 genes in human melanoma A375 cells was inhibited by si RNA interference technology.The effects of CBP and Nrf2 gene silencing on A375 cells were studied,which laid a foundation for the development of novel nucleic acid drugs.Method: 1.One pair of specific CBP si RNA and three pairs of specific Nrf2 si RNA were designed and synthesized for CBP and Nrf2 gene m RNA sequences,and si RNA was transfected into melanoma A375 cells by liposome efficient transfection reagent Lipo High.2.Real-time quantitative PCR was used to detect the m RNA expression levels of CBP and Nrf2 genes after 48 hours of transfection.3.After si RNA transfection,the proliferation ability of A375 cells was detected by CCK-8 kit(Cell Counting Kit 8).4.Beyotime active oxygen kit to determine the ROS content of each group of cells.5.The distribution of CBP and Nrf2 si RNA to A375 cells was determined by PI staining combined with flow cytometry.6.The apoptosis rate of CBP and Nrf2 si RNA on A375 cells was determined by Annexin V-FITC/PI staining combined with flow cytometry.7.The morphology of cells or nuclei was detected by light microscopy and DAPI staining at different time points.Results: 1.The results of Real-time PCR showed that the expression levels of CBP and Nrf2 genes in CBP si RNA and Nrf2 si RNA groups were lower than those in the control group(P<0.05).2.After A375 cells were transfected with different doses of CBP si RNA and Nrf2 si RNA,CCK-8 results showed that after inhibiting CBP and Nrf2 genes,the cell proliferation rate decreased with the increase of si RNA dose.3.After A375 cells were transfected with different doses of CBP si RNA and Nrf2 si RNA,ROS results showed that both CBP si RNA and Nrf2 si RNA increased intracellular ROS content,and the ROS of the co-transfection group was significantly higher than that of the single transfection group.4.CBP si RNA decreased the proportion of G2/M phase(P<0.05);Nrf2 si RNA blocked A375 cell cycle in S phase(P<0.05).Co-transfection of CBP,Nrf2 si RNA resulted in S phase arrest in cells.5.With the increase of CBP si RNA(Nrf2 si RNA)dose,the proportion of normal cells is less,the number of late apoptosis increases,and it has a certain dependence on the transfection dose.6.The morphology of the cells was observed under a light microscope.The cells in the control group and the negative control group grew adherently,and the cell volume was larger.As the culture time increased,the number of cells gradually increased.After 4 hours of transfection with CBP si RNA,the cells showed cytoplasmic foaming,and the number of cells decreased with the increase of culture time.Under confocal fluorescence microscopy,the cells showed nuclear condensation and nuclear degradation after 36 hours of transfection with CBP si RNA.After transfection with Nrf2 si RNA,some cells were shrunk and detached,the intercellular space increased,and the cell morphology was irregular.Under confocal fluorescence microscopy,chromatin accumulation,deep staining and nuclear degradation were observed in cells transfected with Nrf2 si RNA.After co-transfection of CBP and Nrf2 si RNA,the cell morphology was reduced and the cytoplasm was condensed.Under the confocal fluorescence microscope,the nuclear degradation gradually disappeared at 48 h in the co-transfection group.Conclusion:1.CBP si RNA raises ROS in tumor cells,causing cytoplasmic vacuolation.At this time,the cells are not completely dead,but the chromatin is abnormally compressed in the nucleus from the time of transfection with CBP si RNA,eventually leading to cell death.2.Nrf2 si RNA raises ROS in tumor cells and promotes tumor cell death.Mainly by affecting the cell cycle,the tumor cells are arrested in the S phase,reducing the number of cells entering the G2/M phase,thereby inhibiting tumor proliferation.3.Co-transfection of CBP and Nrf2 si RNA mediates the production of ROS,and causes S-phase arrest in cells to promote cell death.CBP is not only involved in the regulation of ROS by the Keap1-Nrf2-ARE pathway,but also participates in other pathways to regulate ROS levels.