HBsAg Inhibits the Translocation of JTB into Mitochondria in HepG2 Cells and Potentially Plays a Role in HCC Progression

Background and Aims: The expression of the jumping translocation breakpoint (JTB) gene is upregulated in malignant liver tissues; however, JTB is associated with unbalanced translocations in many other types of cancer that suppress JTB expression. No comprehensive analysis on its function in human hepatocellular carcinoma (HCC) has been performed to date. We aimed to define the biological consequences for interaction between JTB and HBsAg in HCC cell lines. Methods: We employed the stable transfection to establish small HBsAg expressing HepG2 cell line, and stably silenced the JTB expression using short hairpin RNA in HepG2 cell line. The effects of JTB and small HBsAg in vitro were determined by assessing cell apoptosis and motility. Results: Silencing of JTB expression promoted cancer cell motility and reduced cell apoptosis, which was significantly enhanced by HBs expression. Expression of HBsAg inhibited the translocation of JTB to the mitochondria. Furthermore, silencing of the JTB resulted in an increase in the phosphorylation of p65 in HepG2 cells and HepG2-HBs cells, whereas HBsAg expression decreased the phosphorylation of p65. The silencing of JTB in HepG2-HBs cells conferred increased advantages in cell motility and anti-apoptosis. Conclusion: HBsAg inhibited the translocation of JTB to the mitochondria and decreased the phosphorylation of p65 through the interaction with JTB, After JTB knockdown, HBsAg exhibited a stronger potential to promote tumor progression. Our data suggested that JTB act as a tumor suppressor gene in regards to HBV infection and its activation might be applied as a therapeutic strategy for in control of HBV related HCC development.National Natural Science Foundation of China [30971362, 81072013]; Fundamental Research Funds for the Central Universities in China [2010111082]; Key Projects for Technology Plan of Fujian Province in China [2009D020]; Foundation of Health Bureau of Fujian in China [2007CXB8, 3502z20077046]; Foundation of Health Bureau of Xiamen in China [2007CXB8, 3502z20077046

The Silencing of Pokemon Attenuates the Proliferation of Hepatocellular Carcinoma Cells In Vitro and In Vivo by Inhibiting the PI3K/Akt Pathway

Youth Innovation Fund of Fujian Province [2006F3127]; National Natural Science Foundation of China [30971362, 81072013]; Fundamental Research Funds for the Central Universities in China [2010111082]Pokemon (POK erythroid myeloid ontogenic factor), which belongs to the POK protein family, is also called LRF, OCZF and FBI-1. As a transcriptional repressor, Pokemon assumes a critical function in cellular differentiation and oncogenesis. Our study identified an oncogenic role for Pokemon in human hepatocellular carcinoma (HCC). We successfully established human HepG2 and Huh-7 cell lines in which Pokemon was stably knocked down. We demonstrated that Pokemon silencing inhibited cell proliferation and migration. Pokemon knockdown inhibited the PI3K/Akt and c-Raf/MEK/ERK pathways and modulated the expression of various cell cycle regulators in HepG2 and Huh-7 cells. Therefore, Pokemon may also be involved in cell cycle progression in these cells. We confirmed that Pokemon silencing suppresses hepatocellular carcinoma growth in tumor xenograft mice. These results suggest that Pokemon promotes cell proliferation and migration in hepatocellular carcinoma and accelerates tumor development in an Akt- and ERK-signaling-dependent manner

JTB interaction with HBs in HepG2 cells.

<p>(A) The expression levels of JTB in HCC cell lines and normal liver cell, JTB in gastric cancer cells and normal gastric cell were determined using western blot analysis. HBs was detected for stable transfected cells. β-actin was used as a loading control. (B) Immunoprecipitation was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036914#s2" target="_blank">Materials and Methods</a> section. JTB and HBs were detected by western blot analysis. The whole-cell lysate was used as a positive control. (C) A western blot showing the distribution of JTB in the cytosolic and mitochondrial subfractions. Tubulin and coxIV were used as cytosolic and mitochondrial markers, respectively. (D) The effects of JTB on the mitochondrial localization and co-localization of JTB and HBs. Cellular localization by the immunocytochemical analysis of JTB and HBs in HepG2 cells using (a, b) FITC-labeled anti-JTB (green) and Texas red-labeled anti-coxIV (red) antibodies, (d, e) FITC-labeled anti-JTB (green) and Texas red-labeled anti-HBs (red) antibodies, (g, h) and FITC-labeled anti-JTB FITC (green) and Texas red-labeled anti-coxIV (red) antibodies under confocal microscopy. To label the nuclei, 4′,6-diamidino-2-phenylindole (DAPI) was used (blue). Bars, 10 μm.</p