| ObjectiveTo investigate the effect of Rac1 protein on apoptosis and necroptosis of SK-Hep1 and p~0SK-Hep1 cells and mechanism,and to elucidate the methods of Rac1 regulating apoptosis and necroptosis of hepatoma cells,thus providing a new experimental basis for gene targeting therapy of hepatoma cells.Methods1.SK-Hep1 cells were cultured in vitro,and their mitochondrial DNA-defected cells(p~0SK-Hep1)were induced.The living cells were identified by Oftrypan exclusion and the expression of COX-? and COX-? were detected by RT-PCR.2.Three kinds of necroptosis inhibitors Nec-1(30?M,60?M,90?M)and z-VAD-FMK(10?M,20?M,40?M)were used to detect the optimal concentration of the drug in hepatoma cells after treatment for 24 h.The effects of drugs on apoptosis and necroptosis were detected by flow cytometry.3.The expression of Rac1 in SK-Hep1 and p~0SK-Hep1 cells was down-regulated by adding Rac1 siRNA,and the apoptotic rate and necrotic apoptosis rate were detected by flow cytometry.4.SK-Hep1 cells and p~0SK-Hep1 cells were pretreated with the optimal concentration of necroptosis inhibitor Nec-1 and broad-spectrum apoptosis inhibitor z-VAD-FMK for 24 h.Three siRNAs(FADD,RIP1 and TRAF6)were transfected respectively and their expressions were silenced.The silencing of three factors and the expression of Rac1 at protein and molecular level were detected by RT-PCR and Western blot.Results1.Using Of trypan to identify living cells,the result showed that there was no viable cells at the 6th day in the culture medium containing 100 g·L-1 EB,pyruvate and uracil.But in the medium containing EB,pyruvate and uridine,cells were grown normally and attached,only a small amount were suspended.2.The expressions of COX-?,COX-? and GAPDH were found in SK-Hep1 cells,but not in p~0SK-Hep1 cells(P<0.01).3.Nec-1 and z-VAD-FMK at different concentrations were used to treat SK-Hep1 for24 h.OD values at 490 nm were compared with those in blank group,and cell survival rate was calculated.The cell survival rate was statistical significantly when Nec-1 concentration was 30?M than that in control group(P<0.05).The survival rate of Nec-1 at concentration of 60?M was 104.72%,higher than 92.45%at concentration of 90?M(P<0.05);When z-VAD-FMK concentration was 1O?M,compared with the control group,the cell survival rate was statistical significantly(P<0.05),and the survival rate at the concentration of 20?M,the cell survival rate was 91.10%,higher than the concentration of 40?M with 87.82%(P<0.05).4.The percentages of apoptosis and necroptosis in untreated SK-Hep1 cells were 19.69%and 12.36%respectively,and the apoptotic rate was 21.90%when Nec-1 was added,and the proportion of necroptosis was 25.18%when z-VAD-FMK was added to the cells;And the proportion of apoptosis and necroptosis were 15.38%and 17.52%respectively when Nec-1 and z-VAD-FMK were added to the control group at the same time.The ratios of apoptosis and necroptosis were significantly higher than that of the control group(P<0.05).5.When Rac1 siRNA was transfected into hepatoma cells,RT-PCR assay showed that the expression of Rac1 mRNA decreased significantly(P<0.01).Further,the flow analysis results show that,the apoptotic rates of untreated SK-Hep1 cells and p~0SK-Hep1 cells were 9.37%and 13.04%,respectively;The apoptotic rates of blank control group were 10.01%and 10.18%respectively;The apoptotic rate of SK-Hep1 cells transfected with Ra1 lsiRNA was 3.59%and that of p~0SK-Hep1 cells transfected with Rac1 siRNA was 6.40%.Compared with the blank control group,the difference was statistically significant(P<0.01).6.SK-Hep1 and p~0SK-Hep1 were pretreated with Nec-1 and z-VAD-FMK.After transfections of FADDsiRNA,RIP 1 siRNA and TRAF6siRNA for 48 h,the expressions of FADD,RIP1,TRAF6 were all significantly decreased(P<0.01).And on this basis,the expression of Rac1 in SK-Hep1 cells was decreased after TRAF6 was silencing(P<0.05),but the expressions did not change significantly(P>0.05)after the silencing of FADD and RIP1(P>0.05);While in p~0SK-Hepl cells,the expression of Rac1 also decreased significantly after TRAF6 was silencing(P<0.01),but the expressions of Rac1 did not change either after FADD and RIP1 were silencing(P>0.05).7.After FADD,RIP1 and TRAF6 were transfected,Nec-1 and z-VAD-FMK were added to SK-Hepl cells at the same time.It was found that the expression of Rac1 mRNA in SK-Hepl cells decreased significantly after TRAF6 was silencing(P<0.01),while in p~0SK-Hep1 cells,the expression of Rac1 mRNA was also decreased after TRAF6 was silencing(P<0.05),but after silenced of FADD and RIP1,the expressions of Rac1 mRNA in SK-Hep1 and p~0SK-Hep1 cells were no significant changes(P>0.05).In addition,there was no significant difference in the expressions of Racl mRNA between SK-Hepl and p~0SK-Hepl cells in the results of PCR(P>0.05).Conclusions1.The model of mitochondrial DNA deletion cells(p~0SK-Hepl)was successfully constructed.2.When the process of necroptosis was inhibited,apoptosis will be induced,and when the process of apoptosis is blocked,necroptosis will also be stimulated.3.Racl regulates the process of apoptosis in SK-Hepl cells,and the mechanism may be mediated by TRAF6 to induce apoptosis and necroptosis of hepatoma cells,however,FADD and RIP1 did not play a significant role.4.Mitochondrial DNA deletion has no significant effects on the expression of Rac1 in hepatocellular carcinoma cells. |
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