Effects of AFP gene silencing on apoptosis and proliferation of a hepatocellular carcinoma cell line.

Alpha fetoprotein (AFP) is an oncoembryonal protein that is highly expressed in the majority of hepatocellular carcinomas. Previous studies have shown that AFP may be involved in multiple cell growth regulating, differentiating, and immunosuppressive activities. We investigated the effects of AFP gene silencing by siRNA on apoptosis and proliferation of hepatocellular carcinoma cell line EGHC-9901, which highly expresses AFP and may serve as an ideal model for investigation of AFP functions. siRNA expressing plasmid targeting the AFP gene was first established and subsequently transfected into hepatocellular carcinoma cell line EGHC-9901; cells were then divided into three groups: siRNA-afp, transfected with AFP-siRNA; siRNA-beta-actin, transfected with siRNA-beta-actin as the positive group; and vector control, transfected with empty vector as the blank control group. After G418 positive clone selection for a couple of weeks, Western blot and RT (reverse transcription)-PCR assay demonstrated that AFP expression was almost completely inhibited by siRNA-afp, which indicates that siRNA expressing plasmid targeting the AFP gene has been successfully established. Furthermore, MTT (methyl thiazolyl tetrazelium) assay showed that cells transfected with siRNA-afp proliferated at a significantly lower speed than the other two groups and flat plate clone formation assay also witnessed less clones with diameters of more than 75 μm in siRNA-afp immunofluorescence indicating that the apoptosis rate of cells transfected with siRNA-afp was significantly higher than the other two groups. Furthermore, flow cytometry manifested approximately 20% more cells of siRNA-afp within G1 phase than those of the negative group, indicating that inhibition of AFP expression may cause G1 phase arrest. Finally, Western blot and RT-PCR assay demonstrated that siRNA-afp induced a higher expression of caspase-3 than the other two groups whereas there was no difference in expression of caspase-8, caspase-9, and Bcl-2 between the three groups.