| Objective:In this study,we explore the role of RMP in apoptosis induced by TRAIL and investigate the mechanism of RMP in regulating the apoptosis.Methods:1.The apoptosis of cells treated with cisplatin was measure by flow Cytometry.2.The lentivirus pCDNA3.1-RMPo and pGPU6-RMPi were infected into human hepatoma cells HepG2 for 48-72 hours,and then treated with 500 ng/ml puromycin to obtain stable cell lines.And the relative expression of mRNA and protein of the stable cell lines were detected by fluroscence quantitative PCR and Western blot.3.The stable cell lines were treated with 100 ng/ml TRAIL respectively.After 24 hours,the apoptosis was measured by flow cytometry.4.The stable cell lines were cultured with different concentrations(0 ng/ml,20 ng/ml,40 ng/ml,60 ng/ml,80 ng/ml,100 ng/ml)for 6 hours,and CCK8 was used to detect the toxic effects of different concentrations of TRAIL.5.The stable cell lines were treated with 100 ng/ml TRAIL respectively.After 8 hours,the protein expression of p-Bad,pro-Caspase 3 was detected by Western blot.6.The stable cell lines were treated with 100ng/ml TRAIL respectively.After 3 hours,the mRNA relative expression of Caspase 8 and DR4 was detected by fluroscence quantitative PCR.7.The stable cell lines were treated with 100 ng/ml TRAIL respectively.After 3 hours,the mRNA relative expression of p53 was detected by fluroscence quantitative PCR.8.The stable cell lines were treated with 100 ng/ml TRAIL respectively.After 8 hours,the protein expression of p53 was detected by Western blot.9.The protein of p53 was detected by Western blot after the plasmids of PCDNA3.1-RMPo and pGPU6-RMPi were transiently transfected in HepG2 with different doses(0 ?g,1 ?g,1.5 ?g,2 ?g).10.The pCDNA3.1-RMPo and pCDNA3.1-p53 o plasmid were co-transfected in HepG2 cells,and treated with 100 ng/ml TRAIL for 24 hours,the apoptotic was measured by flow cytometry.11.The plasmid pGL3-basic-p53 promoter was successfully constructed.Fluorescence activity was measured by multifunctional microplate reader to detect the interaction of RMP with the p53 promoter.Resluts:1.TRAIL could induce the apoptosis of HepG2 and MDA-MB-231 cells,but not 7701 and 293 t cells.And also shows HepG2 cells have drug tolerance to TRAIL.2.The stable cell lines of Vector,RMPo and RMPi were successfully established.3.Although apoptosis in all cells increased during the time course after induction with TRAIL,depletion of RMP resulted in more apoptotic cells.However HepG2 cells with RMP overexpression were resistant to TRAIL with less apoptotic cells compared with Vector cells.4.The toxic of TRAIL on cells enhanced with the increasing concentration of TRAIL.However HepG2 cells with RMP overexpression were resistant to TRAIL with less toxic compared with Vector cells.5.The expression of p-Bad was significantly elevated by RMP overexpressed in HepG2,however the activity of Caspase 3 was inhibited in the RMP overexpression cells(RMPo)compared with Vector cells.6.The mRNA relative expression of Caspase 8 and DR4 was significantly reduced by RMP overexpressed in HepG2.There was no significant change in the mRNA expression of Caspase 8 and DR4 before and after TRAIL expression.7.The mRNA relative expression of p53 was significantly reduced by RMP overexpressed in HepG2.There was no significant change in the mRNA expression of p53 before and after TRAIL expression.8.The protein expression level of the p53 was significantly reduced by the increasing amount of RMP.9.The apoptosis rate of HepG2 cells co-transfected with pCDNA3.1-RMPo and pCDNA3.1-p53 o was significantly elevated by flow cytometry compared with untransfected cells.10.RMP can not act on p53 promoter to inhibit p53 transcription.Conclusion:1.RMP can inhibit TRAIL-mediated apoptosis of hepatocellular carcinoma cells,so that liver cancer cells are resistant to TRAIL.2.RMP inhibits TRAIL-mediated apoptosis by inhibiting p-Bad and Caspase 3.3.RMP inhibits the expression of p53 mRNA and protein levels.4.RMP inhibits DR4 and Caspase 8,thereby inhibiting TRAIL-mediated apoptosis.5.RMP does not act on the p53 promoter. |
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