RIPK4 gene silencing enhances TNF-a-induced apoptosis of osteosarcoma MG-63 cells / 肿瘤

ABSTRACT

Objective: To investigate the effect of receptor interacting protein kinase 4 (RIPK4) gene silencing on tumor necrosis factor-a (TNF-a)-induced apoptosis of human osteosarcoma MG-63 cells. Methods: The specific siRNA targeting RIPK4 gene (RIPK4-siRNA) was transfected into MG-63 cells by liposome (named as RIPK4-siRNA group), while the MG-63 cells were transfected with negative control-siRNA (NC-siRNA) as the negative control (named as NC-siRNA group). Then the MG-63 cells transfected with RIPK4-siRNA were treated with human recombinant TNF-a (named as RIPK4-siRNA+TNF-a group), while the MG-63 cells were only treated with TNF-a as the positive control (named as TNF-a group). The proliferation of MG-63 cells was detected by 5-ethynyl-2'-deoxyuridine (EdU) method. The apoptosis of MG-63 cells was detected by Hoechst 33258 staining. The expressions of RIPK4, Bax, Bcl-xL and caspase-3 proteins in MG-63 cells were examined by Western blotting. Results: The expression level of RIPK4 protein in MG-63 cells was significantly down-regulated after transfection of RIPK4-siRNA (P < 0.05). The EdU positive rates of RIPK4-siRNA, NC-siRNA, TNF-a and RIPK4-siRNA+TNF-a groups were 60.7%, 39.6%, 43.3% and 16.7%, respectively. The proliferation of MG-63 cells in RIPK4-siRNA group was lower than that in NC-siRNA group (P < 0.05). Furthermore, the proliferation in RIPK4-siRNA+TNF-a group was lower than those in other three groups (all P < 0.05). The apoptosis of MG-63 cells in RIPK4-siRNA groups was significantly higher than that in NC-siRNA group (P < 0.05). Furthermore, the apoptotic rate in RIPK4-siRNA+TNF-a group was significantly higher than those in other three groups (all P < 0.05). The expression levels of Bax and caspase-3 proteins in RIPK4-siRNA group were significantly higher than those in NC-siRNA group (P < 0.05), but the expression level of Bcl-xL protein in RIPK4-siRNA group was significantly lower than that in NC-siRNA group (P < 0.05). The expression levels of Bax and caspase-3 proteins in RIPK4-siRNA+TNF-a group were significantly increased, but the expression of Bcl-xL protein was significantly decreased as compared with other three groups (all P < 0.05). Conclusion: RIPK4 gene silencing can induce the apoptosis of MG-63 cells, and may increase the sensitivity of apoptosis induced by TNF-a.