Effect Of RIPK4 On Opoptosis In Osteosarcoma Cells And Its Mechanism

Objective: To investigate the effect of siRNA-induced downregulation of receptor interacting protein kinase 4(RIPK 4)gene on apoptosis of human osteosarcoma MG-63 cells.To further investigate the effect of silencing receptor interacting protein kinase 4(RIPK4)by regulating the proliferation and apoptosis of osteosarcoma MG-63 cells through inhibiting AKT/mTOR/Gsk3?/NF-?B signaling pathways.Methods:(1)siRNA targeting RIPK 4 gene(RIPK4-siRNA)were transfected into osteosarcoma MG-63 cell line by liposome method.The proliferation of osteosarcoma MG-63 cells was detected by EdU cell proliferation method.The apoptosis of osteosarcoma MG-63 cells was detected by Hoechst 33258 staining.The expression of RIPK4,Bax,Bcl-xl and caspase-3 in osteosarcoma MG-63 cells were examined by Western blot.(2)The specific siRNA targeting RIPK4 gene(RIK4-siRNA)was transfected into MG-63 cells by liposome named as RIPK4-siRNA group.The MG-63 cells were transfected with negative control siRNA(NC-siRNA)group named as NC-siRNA group.Meanwhile,we established the AKT inhibitor Perifosine(KRX-0401)group and the blank control group.We used Immunofluorescence staining to detect the expression of PI3 K,Akt,mTOR and NF-?B in the MG-63 cells.We used Western blot to detect the expression of RIPK4 protein,PI3K/AKT/mTOR,Gsk3 ? and NF-? B signaling proteins in the MG-63 cells,and also used cell western blot to detect the expression of cycle regulatory proteins p53,p21,anti-apoptosis protein Bcl-2,and pro-apoptotic protein Bad in osteosarcoma MG-63 cells.Results:(1)The expressions of RIPK4 protein were significantly down-regulated by transfecting RIPK4-siRNA into MG-63 cells(P <0.05),the result of EdU cell proliferation analysis showed that the proliferation of RIPK4-siRNA groups were lower than that of control group(P<0.05),Hoechst 33258 staining showed that the apoptosis of RIPK4-siRNA groups were significantly higher than that of the untreated groups(P<0.05),and the apoptotic rate of the cellstreated with 10 ng / ml TNF-? were significantly higher than that of the untreated groups(P <0.05).The expressions of Bax protein and caspase-3 protein in the RIPK4-siRNA group were significantly higher than that in the untreated group(P<0.05),the expressions of Bax protein and caspase-3 protein in the RIPK4-siRNA group were significantly higher than that in the untreated group(P<0.05).The expressions of Bax protein and caspase-3 protein were increased in the cells treated with 10 ng/mL TNF-?,and the expressions of Bcl-x1 protein were decreased compared with that of control group(P<0.05).The expression levels of Bax protein and caspase-3 protein in RIPK4-siRNA group treated with 10 ng/mL TNF-? were significantly higher than that incontrol group(P<0.05),and the expression levels of Bax protein and caspase-3 protein were significantly increased,Bcl-xl protein levels were significantly decreased compared with RIPK4-siRNA groups,the difference was statistically significant(P<0.05).(2)The results of immunofluorescence staining showed that there was no significant change in the expression of PI3 K protein,AKT/mTOR proteins were down-regulated and NF-? B protein was up-regulated after siRNA targeting RIPK4 gene(RIK4-siRNA)was transfected into MG-63 cells by liposome compared with blank control group and NC-si RNA group.Western blot analysis showed that in the RIPK4-siRNA group,the expression of RIPK4 protein was significantly downregulated(P<0.05)and there was no significanly change in the expression of PI3K(P>0.05)compared with blank control group and NC-siRNA group.The expression of Akt,mTOR,NF-? B protein in RIPK4-siRNA transfection group and Akt inhibitor group were significantly down regulated(P<0.05)but the expression of Gsk3 ? was significantly up regulated compared with blank control group and NC-siRNA group.Furthermore,we found that Akt inhibitor Perifosine(KRX-0401)had no significant effect on the expression of RIPK4 protein,and there was no statistical significance(*P>0.05).The expression of Akt protein in the RIPK4 group was up-regulated than the Akt inhibitor Perifosine(KRX-0401)group(*P < 0.05),but the expression of NF-?B protein was significantly down-regulated(P < 0.05).In RIPK4-siRNA group and Akt inhibitor Perifosine(KRX-0401)interference group,the expression of the cell cycle regulatory proteins p53,p21 and pro-apoptosis protein Bad was up-regulated,however,the anti-apoptotic protein Bcl-2 was down-regulated(P < 0.05)compared with the control group and the NC-siRNA group(P < 0.05).Conclusion: Silencing the expression of RIPK 4 gene can induce the apoptosis of MG-63 cells,and may increase the sensitivity of apoptosis induced by TNF-?.Silencing RIPK4 gene may regulate the proliferation and apoptosis of osteosarcoma MG-63 cells through AKT/mTOR/Gs3k?/NF-?B signaling pathways.