Effect Of Sirna-mediated Inhibition Of Nuclear Transcription Factor-κb On Apoptosis Of Hepatocellular Carcinoma Cells

Objective:Hepatocellular carcinoma (HCC) is one of the most frequent malignant tumors worldwide, the vast majority is attributed to hepatitis viruses (HBV, HCV) infection.Nuclear transcription factor (NF)-κB can be stitutively highly expressed in cancer cells and promotes tumorogenicity, both inhibiting apoptosis and stimulating proliferation. Although constitutive NF-κB activity has been implicated in HCC,the relation between HBV replication and NF-κB activation is not clear, and the role that NF-κB plays in HCC pathogenesis needs to be futher elucidated. The aim of this study was to investigate the expression of NF-κB in different tissues of HBV-related HCC and its correlations with the clinicopathological features,and analyze the effect of siRNA-mediated inhibition of NF-κB on apoptosis of HepG2 cells.Methods:Using the immunohistochemistry, NF-κB expression was detected in the tissues of 35 HCC and the corresponding non-cancerous tissues.Liver HBV-DNA was detected by in situ molecular hybridization technique.The relationships between NF-κB expression and HBV replication or clinical pathological characteristics were analyzed. siRNA was used to down-regulate NF-κB expression in HepG2 cells,qualitative and quantitative PCR, ELISA and Western blot were used to examine NF-κB expression; Annexin V-FITC was used to test cell apoptosis.Results:The positive NF-κB material was brown granule-like staining, the NF-κB with dot-nest-like staining localized both in nucleus and cytoplasm in HCC,and only in cytoplasm in its surrounding tissues.Its expression in HCC was well-distributed and stronger than in its surrounding tissues.The incidence of NF-κB positive expression was 100% in HCC and 68.6% in its surrounding tissues,respectively. Significant difference was found between two groups (Fisher's value= 0.000).No positive relationship presented between NF-κB expression and histological differentiation grade, the number of tumor, the size of tumor, the status of HBsAg and AFP level.The NF-κB expression in HBV-DNA-positive HCC group was significantly higher than that in HBV-DNA-negative one (t=4.7347,P=0.000).72 h after siRNA transfection, HepG2 cells NF-κB expression in 100 nM group reduced 93% and 62% at the mRNA and protein levels,respectively. The down-regulation of NF-κB expression was depended on the dose of siRNA and the time after transfection. The apoptosis of HepG2 cells increased 85% with NF-κB inhibition.Conclusion:The abnomal activation of hepatic NF-κB is closely associated with the occurrence and development of HCC, and NF-κB inhibition mediated by siRNA promotes HepG2 cells apoptosis. Therefore NF-κB is a potential target for HCC gene therapy.