Study On The Roles Of CBP,NOX2,Ku70 And Mre11 In Cell Death Of Human Melanoma

Objection:Explore the mechanism of gene transcription regulation network composed of CBP,NOX2,Ku70,and Mre11 to regulate the apoptosis,paraptosis and necrosis in human melanoma cells,targeting siRNA for the discovery of the combined regulation of ROS and DNA damage repair mechanisms to treat melanoma,which laid a founction for the development of molecular mechanisms of drugs and diseases caused by CBP deficiency.Method:1.Three pair of specific Ku70 siRNAs and three pair of specific Mre11 siRNAs were designed and synthesized for Ku70 and Mre11 gene mRNA sequences,and siRNA was transfected into melanoma A375 cells by liposome efficient transfection reagent Expect2000.2.Real-time quantitative PCR was used to detect the mRNA expression levels of CBP,Ku70 and Mre11 genes after 24/48 hours of transfection.70% knockdown efficiency was used as the working dose for CBP siRNA and Ku70 siRNA,CBP siRNA and Mre11 siRNA single transfection and co-transfection.3.After transfection for 24 h,the proliferation and death of A375 cells was detected by CCK-8 and flow cytometry.4.After transfection for 24 h,the morphological changes and ROS level of A375 cells was detected by flow cytometry.5.RT-qPCR and Western Blot were used to detect the expression of NOX2 in cells after 24 hours of transfection.6.The morphology of cells or nucleus was detected by light microscopy and DAPI staining at different time points.7.After transfection for 24 h,RT-qPCR and Western Blot were used to detect the expression of Ku70.8.After transfection for 24 h,the distribution of A375 cells were determined by PI staining combined with flow cytometry.9.The experiments were repeated 3 times independently,and the results were statistically analyzed using SPSS22.0.P <0.05 was considered statistically significant.Results:1.CBP and Ku70 siRNA down-regulate the expression of CBP,Ku70 mRNA in melanoma A375 cells in a dose-dependent manner(P <0.05).The knockdown efficiency of 3 fmol/cell CBP siRNA and 2.5 fmol/cell Ku70 siRNA both were 70%.In A375 cells,deplition of CBP and/or Ku70 inhibited the proliferation and caused death of apoptosis,necrosis,and paraptosis in a dose-dependent manner(P <0.05).At 70% knockdown efficiency,there was no significant difference between the effects of CBP siRNA transfection group and CBP-Ku70 siRNA cotransfection group on A375 cell death(P>0.05).Deplition of CBP and/or Ku70 caused cytoplasmic foaming of A375 cells.Deplition of CBP and/or Ku70 lead to an increased level of ROS(P<0.05).Deplition of CBP and/or Ku70 up-regulated the expression of NOX2(P<0.05).Deplition of CBP and/or Ku70 caused nuclear degradation of A375 cells.Deplition of CBP down-regulated the expression of Ku70(P<0.05).Deplition of CBP and/or Ku70 caused A375 cell cycle arrest at S phase.2.Mre11 siRNA down-regulate Mre11 mRNA expression in melanoma A375 cells in a dose-dependent manner(P <0.05).The knockdown efficiency of both 3 fmol / cell Mre11 siRNA and 3 fmol / cell CBP siRNA was 70%.In A375 cells,deplition of Mre11 lead to an increased level of the ROS,inhibited proliferation,and caused death of apoptosis and necrosis(P <0.05).At 70% knock-down efficiency,the apoptosis rate of the CBP-Mre11 siRNA group showed an additive effect compared with the CBP siRNA group(P <0.05).At 70% knockdown efficiency,the nuclear degradation of CBP-Mre11 siRNA was more severe than the CBP siRNA group.At 70% knock-down efficiency,the CBP-Mre11 siRNA group had an additive effect on the S-phase cell block compared with the CBP siRNA group(P <0.05).The difference of ROS level between CBP siRNA group and CBP-Mre11 siRNA group has no statistically significant(P> 0.05).Conclusion:In this study,we found that CBP worked with Ku70 modulated the expression of NOX2.Deplition of CBP and/or Ku70 up-regulated the expression of NOX2,leading to an increased level of ROS.The increased ROS made consistent with the phenomenon of cytoplasmic vacuolization,cell cycle arrested at S phase,caused cell death of paraptosis and necrosis.In addition,we found that CBP plays a double role in both expression and acetylation of Ku70.Deplition of CBP down-regulated the expression of Ku70,thus decrased the number of Ku70-BAX complex,therefore still leading to an amount of free BAX increased to result in cell death of apotosis.These results indicated that CBP,Ku70,and NOX2 consist a gene regulatory network that regulates cell death of paraptosis,necrosis,and apoptosis in human melanoma.In human melanoma,Mre11 and CBP regulated ROS in the same pathway.Deplition of Mre11 can increased the level of ROS,which caused cell cycle arrest at S-phase and inhibited proliferation,thus caused cell death of necrosis that characterized by cytoplasmic foam;Mre11 plays an important role in DNA damage repair,deplition of Mre11 casued cell death of apotosis.In addition,Mre11 is involved in repairing DNA damage caused by CBP deficiency in melanoma cells.