Effects Of Rac1 And IRAK1 On Apoptosis And Necroptosis Of Hepatocellular Carcinoma Cells

ObjectiveBy inhibiting cell apoptosis or necroptosis pathway,to detect the expression of Rac1 and IRAK1 protein in the hepatocellular carcinoma cell SK-Hep1 and the hepatocellular carcinoma cell that mitochondrial DNA deletion P0SK-Hep1,to explore the effect of Rac1 and IRAK1 on the cell apoptosis and proliferation through what kind of Cell death way and the effect of mitochondrial DNA deletion.Methods1.Culturing human hepatocellular carcinoma cell lines SK-Hepl and establishing the hepatocellular carcinoma cell lines P0SK-Hepl that mitochondrial DNA deletion.2.Hepatocellular carcinoma cells SK-Hep1 were treated with different concentrations of specific necroptosis inhibitor Nec-1 and broad spectrum caspase inhibitor z-VAD-FMK respectively,to detect the toxic effect of the drugs on the cells by MTS method and select the appropriate concentration.3.Hepatocellular carcinoma cells SK-Hepl were transfected with FAM-siRNA,fluorescent microscope was used to observed the transfection effect,to select the appropriate transfection conditions for subsequent experiments and ensure that the interference experiment to achieve better interference effect.4.SK-Hepl were treated with Nec-1,z-VAD-FMK and Nec-1 combined with z-VAD-FMK respectively,three different treatment groups were transfected separately three small interfering RNAs associated with apoptotic or necroptosis proteins,extracted the groups of cellular protein,.Western Blotting show that the expression of interfered target protein and Rac1 and IRAK1 protein.5.P0SK-Hepl were treated with Nec-1,z-VA-D-FMK and Nec-1 combined with z-VAD-FMK respectively,three different treatment groups were transfected separately three small interfering RNAs associated with apoptotic or necroptosis proteins,extracted the groups of cellular protein,Western Blotting show that the expression of interfered target protein and Rac1 and IRAK1 protein.Results.1.Treatment of hepatocellular carcinoma cell SK-Hep1 with different concentrations of Nec-1 compared with DMSO group,the cell viability of the 30?M group was different significantly(P<0.05),while the cell survival rate in the 60?M group was higher than that in the 90?M group.2.Treatment of hepatocellular carcinoma cell SK-Hepl with different concentrations of z-VAD-FMK,compared with DMSO group,the cell viability of the 10?M group was different significantly(P<0.05),while the cell survival rate in the 20?M group was higher than that in the 40?M group.3.FAM-siRNA was transfected into hepatocellular carcinoma cell SK-Hepl,under the fluorescence microscope,the transfected hepatocellular carcinoma cell could be seen most of the expression of green fluorescence in blue light excitation,scattered in the cytoplasm.4.In the interference RIP1 of SK and P0SK,compared with the blank group and the negative control group,the expression of Rac1 protein in these three groups was significantly decreased after Nec-1,z-VAD-FMK and Nec-1 combined with z-VAD-FMK(P<0.05).Compared with blank control group and negative control group,the expression of IRAK1 protein was significantly increased after Nec-1 combined with z-VAD-FMK(P<0.05)in SK cells,there was no significant difference in the expression of IRAK1 protein in three groups of P0SK cells(P<0.05).5.In the interference FADD of SK and P0SK,compared with the blank group?the negative control group and Nec-1 group,the expression of Rac1 protein in these three groups was significantly increased after z-VAD-FMK and Nec-1 combined with z-VAD-FMK(P<0.05).Compared with blank control group and negative control group,the expression of IRAK1 protein was significantly decreased after Nec-1 combined with z-VAD-FMK(P<0.05)in SK cells,there was no significant difference in the expression of IRAK1 protein in three groups of P0SK cells(P<0.05).6.In the interference TRAF6 of SK and P0SK,compared with the blank group?the negative control group and Nec-1 group,the expression of Ral protein in these three groups was significantly decreased after z-VAD-FMK and Nec-1 combined with z-VAD-FMK(P<0.05).There was no significant difference in the expression of IRAK1 protein between the three groups(P<0.05).Conclusion1.Rac1 and IRAK1 was expressed in human hepatocellular carcinoma cell lines SK-Hepl and hepatocellular carcinoma cell lines P0SK-Hepl.2.Rac1 and IRAK1 can promote the growth and proliferation of hepatocellular carcinoma cells,and inhibit the cell apoptosis.3.Rac1 mainly affects the proliferation and apoptosis of hepatocellular carcinoma cells through the pathways of apoptosis,unrelated to necroptosis.IRAK1 can affect the proliferation and apoptosis of hepatocellular carcinoma cells through the pathways of apoptosis and necroptosis.4.The deletion of mitochondrial DNA in hepatocellular carcinoma cells had no significant effect on Rac1 induced apoptosis,but had effect on IRAK1 induced apoptosis or necroptosis.