Effects Of RMP And P53 On DNA Damage Repair And Apoptosis In Hepatocellular Carcinoma Cells

ObjectiveSince The hepatocellular carcinoma cells have irradiated tolerance,studying the relationship of RMP and p53 in irradiation induced hepatocellular carcinoma cells contributes to the understanding mechanism of RMP in the hepatocellular carcinoma cells,which provides a possible method to solve the irradiation tolerance of hepatocellular carcinoma therapy.Methods1.The degree of DNA damage in normal liver cells 7701 was detected by comet electrophoresis.2.The protein expression levels of RMP,p53 and phosphorylated p53 in irradiation induced normal liver cells 7701 and hepatocellular carcinoma cells Hep G2 were detected by Western Blot.3.The m RNA and protein expression levels of RMP and p53 in hepatocellular carcinoma cells Hep G2,Huh7,Hep3 B were detected by fluorescence quantitative PCR and Western Blot.4.Fluorescence quantitative PCR and Western Blot were used to detect the m RNA and protein expression leves of RMP and p53 in hepatocellular carcinoma cells Hep G2,Huh7 and Hep3 B transfected with RMP overexpression plasmid and RMP interference plasmid.5.Comet electrophoresis was used to detect DNA damage degree of irradiation induced hepatocellular carcinoma cells Hep G2 and Hep3 B which was transfected with no-load physique,RMP plasmid,p53 plasmid,a total of RMP plasmid and p53 plasmid after 1 hour.6.Flow Cytometer was used to detect cell apoptosis of irradiation induced hepatocellular carcinoma cells Hep G2 and Hep3 B which was transfected with no-load physique,RMP plasmid,p53 plasmid,a total of RMP plasmid and p53 plasmid after 1 hour.7.Western Blot was used to detect the protein expression levels of RMP,p53,Bcl-xl,Bad,p-Bad,?-H2 AX in hepatocellular carcinoma cells Hep G2 and Hep3 B which was transfected with no-load physique,RMP plasmid,p53 plasmid,a total of RMP plasmid and p53 plasmid.Results1.The DNA damage of irradiation induced normal hepatocyte 7701 irradiated by comet electrophoresis showed a significant increase after 1hour.2.Western Blot detected no significant changes in RMP protein expression level after the normal liver cell 7701 irradiation,while its level reached the maximum in hepatocellular carcinoma cells Hep G2 after1 hour irradiation.The protein expression levels of p53 decreased and the protein expression levels of phosphorylated p53 increased.in normal liver cells 7701 and hepatocellular carcinoma cells Hep G2.3.Fluorescence quantitative PCR and Western Blot showed that the m RNA expression levels and protein expression leves of RMP in Huh7(p53 mutant)were less than that of RMP in Hep G2(p53 wild-type).The m RNA and protein expression levels of RMP in Hep3B(p53 deleted)were higher than that of RMP in Hep G2.4.Fluorescence quantitative PCR and Western Blot showed that the m RNA and protein expression levels of p53 in hepatocellular carcinoma cells Huh7 transfected with RMP overexpression plasmid were significantly increased compared with that in the control group,while the results were reverse in RMP interference plasmid.There is no p53 in hepatocellular carcinoma cells Hep3 B transfected with RMP overexpression plasmid and RMP interference plasmid.5.The comet electrophoresis showed that the values of OTM in hepatocellular carcinoma cells Hep G2 and Hep3 B transfected with RMP overexpression plasmid were significantly decreased compared with that in the control group,while the results were reverse in RMP interference plasmid.The values of OTM in hepatocellular carcinoma cells Hep G2 transfected with p53 overexpression plasmid were significantly increased compared with that in the control group,while the results were reverse in p53 interference plasmid.The values of OTM in hepatocellular carcinoma cells Hep3 B transfected with p53 overexpression plasmid were significantly increased compared with that in the control group.The values of OTM in hepatocellular carcinoma cells Hep G2 and Hep3 B transfected with RMP overexpression plasmid and p53 overexpression plasmid were significantly decreased compared with that in the p53 interference plasmid group,which were increased compared with that in the RMP overexpression plasmid group.The values of OTM in hepatocellular carcinoma cells Hep G2 and Hep3 B transfected with RMP interference plasmid and p53 overexpression plasmid were significantly increased compared with that in the RMP interference plasmid group and p53 overexpression plasmid group.6.Flow Cytometer showed that the numbers of cell apoptosis in hepatocellular carcinoma cells Hep G2 and Hep3 B transfected with RMP overexpression plasmid were significantly decreased compared with that in the control group,while the results were reverse in RMP interference plasmid.The numbers of cell apoptosis in hepatocellular carcinoma cells Hep G2 transfected with p53 overexpression plasmid were significantly increased compared with that in the control group,while the results were reverse in p53 interference plasmid.The numbers of cell apoptosis in hepatocellular carcinoma cells Hep3 B transfected with p53 overexpression plasmid were significantly increased compared with that in the control group.The numbers of cell apoptosis in hepatocellular carcinoma cells Hep G2 and Hep3 B transfected with RMP overexpression plasmid and p53 overexpression plasmid were significantly decreased compared with that in the p53 interference plasmid group,which were increased compared with that in the RMP overexpression plasmid group.the numbers of cell apoptosis in hepatocellular carcinoma cells Hep G2 and Hep3 B transfected with RMP interference plasmid and p53 overexpression plasmid were significantly increased compared with that in the RMP interference plasmid group and p53 overexpression plasmid group.7.Western Blot showed that the protein expression of ?-H2 AX in hepatocellular carcinoma cells Hep G2 and Hep3 B transfected with RMP overexpression plasmid were significantly decreased compared with that in the control group,which is similar in p53 interference group.The protein expression of Bcl-xl and phosphorylated Bad in hepatocellular carcinoma cells Hep G2 and Hep3 B transfected with RMP overexpression plasmid were significantly increased compared with that in the control group.That was similar in Hep G2 and Hep3 B transfected with RMP and p53 plasmid.Conclusion1.The protein expressions of RMP in normal liver cells and hepatocellular carcinoma cells are different,and the protein expression levels of RMP in hepatocellular carcinoma cells are increased after irradiation.2.RMP suppresses the expression of p53 each other in hepatocellular carcinoma cells without irradiation.3.P53 promotes cell apoptosis and DNA damage of irradiation-induced hepatocellular carcinoma cells without RMP.4.P53 can remove the suppression of cell apoptosis and DNA damage by RMP.5.RMP suppresses cell apoptosis and DNA damage of irradiation-induced hepatocellular carcinoma cells without p53.