NF-κB silencing combined with matrine-induced apoptosis in hepatocellular carcinoma cells and expression of related molecules

Abstract

Objective To investigate the apoptosis in hepatocellular carcinoma cells induced by NF-κB silencing combined with matrine(MT) and the expression of related molecules.Methods The recombinant eukaryotic expression plasmid NF-κB/P65 siRNA was constructed and transfected into HepG2 cells.RT-PCR was used to detect the efficiency of the constructed RNAi in silencing NF-κB/P65,and the steadily transfected clones of NF-κB/P65 RNAi were selected.The cultured HepG2 cells was randomly divided into four groups: control group,MT group(1.5 g/L),steadily transfected group and combination group(steadily transfected cells+MT).The apoptosis of carcinoma cells was analyzed by flow cytometry;expression of NF-κB/P65,CD95(Fas),Smac,and Survivin mRNA and protein in carcinoma cells was examined by RT-PCR and Western blotting analysis.Results The expression of NF-κB/P65,CD95(Fas),Smac,and Survivin mRNA and protein in MT group was significantly increased compared with that in the control group(P0.05).The expression of CD95 and Smac in the steadily transfected group was significantly higher than that in the control group(P0.05),and the expression of NF-κB/P65 and Survivin was significantly suppressed compared with the control group and the MT group(P0.05).The expression of CD95 and Smac in the combination group was significantly increased compared with that in the other three groups(P0.05),and the expression of NF-κB/P65 and Survivin was significantly lower than that in the MT group(P0.05).The apoptosis rates of the HepG2 cells in the control group,MT group,steadily transfected group,and combination group were 3.21%,6.25%,11.82%,and 21.06%,respectively,with significant difference found between different groups(P0.05). Conclusion The apoptosis in hepatocellular carcinoma cells induced by NF-κB RNAi combined with matrine may be related to increased CD95 and Smac expression and decreased NF-κB/P65 and Survivin expression.