Effects of IL-12 combined with GM-CSF on apoptosis of H22 hepatoma cells

Abstract

Objective To investigate the effects of granulocyte macrophage colony-stimulating factor (GM-CSF) combined with interleukin-12 (IL-12) genes on apoptosis of hepatoma cells. Methods The hepatoma cell lines were cultured in vitro and were divided into four groups: GM-CSF transfection group, IL-12 transfection group, GM-CSF and IL-12 co-transfection group, negative control group (empty load group), respectively.The PIB-CMV3-GM-CSF and PIB-CMV3-IL-12 eukayotic expression vector was built, and 36 h after transfection, fluorescence microscope was used to detect the transfection effect; the expression level of IL-12, GM-CSF, p53, p38 and C-JUN mRNA were detected by RT-PCR, and Western blot was used to examine the expression level of IL-12, GM-CSF, p53, p38 and C-JUN protein.In addition, the flow cytometry was applied to detect cell apoptosis. Results Through fluorescence microscope, green fluorescence was observed in cells of GM-CSF transfection group, IL-12 transfection group, GM-CSF and IL-12 co-transfection group, indicating that the plasmid has successfully transferred into cells.In addition, the expression of p53mRNA in empty load group, GM-CSF transfection group, IL-12 transfection group, GM-CSF and IL-12 co-transfection group were 1.2±0.10, 4.3±0.98, 4.2±0.34, 9.2±0.87, and the protein expression were 1.0±0.10, 3.6±0.34, 3.8±0.30, 5.0±0.60.Compared with the empty load group, the expression level of p53 mRNA and protein were significantly increased in the three plasmid transfection groups (P<0.01). The expression of p53 mRNA and protein were significantly increased in co-transfection group than GM-CSF group and IL-12 group (P<0.01), while in the comparison with GM-CSF transfection group and IL-12 transfection group, the expression level of p53mRNA and protein in the co-transfection group could be improved to a higher degree(P<0.01). Meanwhile, p38 C-JUN mRNA expression levels in empty load group, GM-CSF transfection group, IL-12 transfection group, GM-CSF and IL-12 co-transfection group were as follows: 7.5± 0.9, 3.5±0.45, 3.7±0.25, 1.0±0.11, while p38protein expression levels were 10.1±1.03, 6.1± 0.67, 7.1 ± 0.61, 1.0 ± 0.12, respectively, C-JUN mRNA expression levels were 11.2 ± 1.20, 4.1 ± 0.19, 3.3 ± 0.30, 1.0 ± 0.01, separately, C-JUN protein expression levels were 2.25 ± 0.2, 1.8 ± 0.13, 1.4 ± 0.12, 1.0 ± 0.09.P38, C-JUN mRNA and protein levels were significantly reduced in the three plasmid transfection groups compared with the empty load group (P<0.01). The expression of p38, C-JUN mRNA and protein were reduced to a lower degree in co-transfection group than in GM-CSF transfection group and IL-12 transfection group (P<0.01). Flow cytometer showed that the hepatoma cell apoptosis rate of the empty load group, GM-CSF transfection group, IL-12 transfection group, co-transfection group were (3.43±0.9)%, (5.87±1.02)%, (7.32±1.1)%, (17.47±2.11)%, the rates of the three plasmid transfection groups were significantly higher than that of the empty load group (P<0.01). And the apoptosis rate was significantly increased in the co-transfected group compared with other plasmid groups (P<0.01). Conclusion The combination of GM-CSF and IL-12 could significantly accelerate the apoptosis of hepatoma cells by up-regulating the expression of p53, and down-regulating the expression of p38 and C-JUN. Key words: Hepatoma cell; Macrophage colony-stimulating factor; Interleukin-12; Cell apoptosis