Aims: To explore the biological roles of alpha-fetoprotein (AFP), a tumor-associated antigen in human hepatocellular carcinoma (HCC).
Materials and methods: After knockdown of AFP in HepG2 cells by transfection of specific Stealth™ RNAi, the expression of AFP were detected by reverse transcription polymerase chain reaction at mRNA level and by enzyme-linked immunosorbent assay at the protein level. Then, the effect of silenced AFP on cell proliferation was assessed by dimethylthiazolyl-2,5-diphenyl-tetrazolium bromide assay, and apoptosis assessment with Hoechst33258 and flow cytometry (double stain with fluorescein isothiocyanate/propidium iodide), the roles of AFP in the cell cycle regulation were assessed by flow cytometry. We also detected the expression of some key proteins related to apoptosis pathway by Western immunoblot analysis.
Results: After the transfection for 48 h, the expression of AFP gene was almost abolished, the cell proliferation was inhibited by 47.61%, the number of cells undergoing early apoptosis was significantly increased to 59.47%; cell cycle was arrested with the increase of G0/G1 phase cells from 45.3% to 58.4%. Inhibition of AFP expression also results in decreasing of transforming growth factor-β (TGF-β), mutant P53 expression, and increasing of Bax/Bcl-2 ratio, activation of caspase-3.
Conclusions: The results suggest that AFP may positively regulate cell proliferation by enhancing the apoptosis resistance via effect on TGF-β and p53/Bax/caspase-3 signaling pathway in HepG2 cells. As such, the knockdown of AFP gene should be further investigated in vivo as a novel approach to HCC treatment.
Keywords: Alpha-fetoprotein; RNA interference; apoptosis; hepatocellular carcinoma; transforming growth factor-β.