Increased ATG5-ATG12 in hepatitis B virus-associated hepatocellular carcinoma and their role in apoptosis

AIM To investigate autophagy-related genes, particularly ATG12, in apoptosis and cell cycle in hepatitis B virus(HBV)-associated hepatocellular carcinoma(HCC) and non-HBV-HCC cell lines.METHODS The expression of autophagy-related genes in HBVassociated hepatocellular carcinoma and non-HBV-HCC cell lines and human liver tissues was examined by quantitative real-time reverse transcriptase-polymerase chain reaction(q RT-PCR) and western blotting. The silencing of target genes was used to examine the function of various genes in apoptosis and cell cycle progression. RESULTS The expression of autophagy related genes ATG5, ATG12, ATG9 A and ATG4 B expression was analyzed in Hep G2.2.15 cells and compared with Hep G2 and THLE cells. We found that ATG5 and ATG12 m RNA expression was significantly increased in Hep G2.2.15 cells compared to HepG 2 cells(P < 0.005). Moreover, ATG5-ATG12 protein levels were increased in tumor liver tissues compared to adjacent non-tumor tissues mainly from HCC patients with HBV infection. We also analyzed the function of ATG12 in cell apoptosis and cell cycle progression. The percentage of apoptotic cells increased by 11.4% in ATG12-silenced Hep G2.2.15 cells(P < 0.005) but did not change in ATG12-silenced HepG 2 cells under starvation with Earle's balanced salt solution. However, the combination blockade of Notch signaling and ATG12 decreased the apoptotic rate of HepG 2.2.15 cells from 55.6% to 50.4%(P < 0.05). CONCLUSION ATG12 is important for HBV-associated apoptosis and a potential drug target for HBV-HCC. Combination inhibition of ATG12/Notch signaling had no additional effect on HepG 2.2.15 apoptosis.

Delta-like ligand 4 in hepatocellular carcinoma intrinsically promotes tumour growth and suppresses hepatitis B virus replication

AIM To investigate the role of Delta-like ligand 4(DLL4) on tumour growth in hepatitis B virus(HBV)-associated hepatocellular carcinoma(HCC) in vivo.METHODS We suppressed DLL4 expression in an HBV expressing HCC cell line, HepG2.2.15 and analysed the growth ability of cells as subcutaneous tumours in nude mice. The expression of tumour angiogenesis regulators, VEGF-A and VEGF-R2 in tumour xenografts were examined by western blotting. The tumour proliferation and neovasculature were examined by immunohistochemistry. The viral replication and viral protein expression were measured by quantitative PCR and western blotting, respectively.RESULTS Eighteen days after implantation, tumour volume in mice implanted with sh DLL4 HepG2.2.15 was significantly smaller than in mice implanted with control HepG2.2.15(P < 0.0001). The levels of angiogenesis regulators, VEGF-A and VEGF-R2 were significantly decreased in implanted tumours with suppressed DLL4 compared with the control group(P < 0.001 and P < 0.05, respectively). Furthermore, the suppression of DLL4 expression in tumour cells reduced cell proliferation and the formation of new blood vessels in tumours. Unexpectedly, increased viral replication was observed after suppression of DLL4 in the tumours.CONCLUSION This study demonstrates that DLL4 is important in regulating the tumour growth of HBV-associated HCC as well as the neovascularization and suppression of HBV replication.