The aim of this study was to confirm the localization of recombinant pGPC3+afp-EGFP which expressed a new re-anchored protein named GPC3+afp-EGFP on the cytoplasmic membrane and to investigate its functions against hepatocellular carcinoma (HCC). EGFP expression in transfected HepG2 cells was observed using fluorescence and a confocal microscope. pGPC3+afp-EGFP expression was detected in membranous and soluble proteins extracted from transfected human embryonic kidney 293 cells by Western blot analysis using GPC3 mAb. The proliferation of transfected HepG2 cells with pGPC3+afp-EGFP (experimental group) was detected using SRB assay and compared to those of transfected HepG2 cells with pGPC3 (control group) and non-transfected HepG2 cells (blank group). Quantitative analysis of mRNA expression of the Fas gene was conducted by real-time PCR using the β-actin housekeeping gene as the internal control at variable times. Apoptotic HepG2 cells in the three groups were counted and statistically analyzed by a contingency table Chi-square test using Spss 11.5 software and TUNEL assay. Production of both TNF-α and IFN-γ/IL2 was detected by ELISPOT after co-cultivation of transfected HepG2 cells with peripheral blood lymphocytes at different time-points in the experimental group. Green fluorescence was mainly found around the transfected HepG2 cell periphery through fluorescence and confocal microscopy. GPC3+afp-EGFP could not be detected in soluble protein but only in membranous protein. Proliferation curves showed that the proliferative quantities of transfected HepG2 cells in the experimental group decreased, whereas the mRNA expression of the Fas gene increased significantly compared to those of the other two groups. The numbers of apoptotic cells in the experimental group were significantly higher compared to those in the other two groups, as shown by statistical analysis. Both TNF-α and IFN-γ/IL2 were induced and were much higher in the experimental groups than in the diverse control groups at variable times. A new re-anchored protein GPC3+afp-EGFP expressed by recombinant pGPC3+afp-EGFP was localized on the cytoplasmic membrane, and had multiple functions against HCC, such as inhibition of transfected HepG2 cell proliferation, promotion of transfected HepG2 apoptosis and induction of antitumor cytokine excretion.

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