WISP-1: a novel mediator of hepatitis B virus-related hepatocellular carcinoma / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the effect of hepatitis B virus (HBV) X protein (HBX) on expression of the host gene Wnt induced secreted protein-1 (WISP-1) that is related to the pathogenic process of hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Tumor and paratumor tissues were collected from HCC patients, and normal liver tissues were collected from healthy controls. Immunohistochemistry was used to evaluate the in vivo presence and expression levels of HBX and WISP-1 in the three tissue types. HepG2 cells stably transfected with pc-DNA3.1(+)-HBX or with pc-DNA3.1(+) only (G0, control) were generated and used to examine in vitro the HBX-induced changes in WISP-1 expression at the mRNA and protein levels by reverse transcription polymerase chain reaction and western blotting, respectively.</p><p><b>RESULTS</b>The HCC tissues showed significantly higher rates of positivity for WISP-1 expression than the non-tumor controls (76.6% vs. paratumor: 23.4% or normal tissues: 0%, x2= 35.967, P less than 0.01). HBX increased WISP-1 expression in HepG2 cells at both the mRNA (1170.33 +/- 41.26 vs. G0: 265.34 +/- 27.47, t = 31.63, P less than 0.01) and protein (240.33 +/- 11.37 vs. G0: 40.33 +/- 7.09, F = 600.57, P less than 0.01) levels.</p><p><b>CONCLUSION</b>HBV may up-regulate expression of the host gene WISP-1 through its X protein and thus promote the development of HCC.</p>

The effect of hepatitis B virus X protein on the expression of CtIP in HepG2 Cells / 中华肝脏病杂志

To investigate the effect of hepatitis B virus X protein(HBx) on CtBP-interacting protein(CtIP) which is an important repair factor of DNA double strand break damage in HepG2 cells induced by bleomycin. A HBx stably expressing HepG2 cell line and a control HepG2 cell line with empty vector transfected were established. After the double strand break (DSB) damage occurred, the mRNA and protein levels of CtIP were detected by Real-time PCR and Western blot assay respectively, cell cycle profiles and apoptotic cell death were determined by a flow cytometry, and the position of CtIP in cells was observed by confocal laser scanning microscopy. It showed that HepG2 cells transfected with hepatitis B virus X gene could stably express HBx protein. After being induced by bleomycin, the percentage of apoptotic cell was 16.90%+/-0.89% in HBx stably expressing HepG2 cell line and 15.30%+/-0.86% in control cell line, respectively (q = 2.074, P is more than to 0.05). While the percentage of death cell was 8.71%+/-0.74% in HBx stably expressing HepG2 cell line and 4.90%+/-0.46% in control cell line, respectively (q = 7.126, P is less than to 0.01). The two cell lines manifested the increase of G2/M arrest and significant difference existed between the two cell lines. HBx down regulated the expression levels of CtIP and its mRNA. The CtIP level was 0.66+/-0.04 in HepG2-HBx cell and 0.73+/-0.05 in HepG2-vec cell, respectively (t = 2.314, P is less than to 0.05). The relative mRNA level was 1.00+/-0.06 in HepG2-HBx cell and 1.23+/-0.08 in HepG2-vec cell, respectively (t = 2. 732, P is less than to 0.05). We also found that CtIP was concentrated in the cell nucleus. The research suggests that HBx may affect DNA-repair pathways by disrupting the expression of CtIP.

HBx gene down-regulates miR-192 expression and inhibits apoptosis of human hepatoma cell line HepG2 / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the mechanism by which HBV X gene(HBx) inhibits apoptosis of human hepatoma cell line HepG2 in terms of miRNA.</p><p><b>METHODS</b>Three cell lines were prepared: HepG2 cells stably transfected with HBx (HepG2/HBx), HepG2 cells stably transfected with pcDNA3.1 (HepG2/pcDNA3.1) and HepG2 cells. Flow cytometry was adopted to measure the apoptosis of these three cells and Taqman fluorescence quantitative PCR was used to examine miR-192 expression. After HepG2 cells was transfected with miR-192, the apoptosis was analyzed by flow cytometry and the expressions of p53 and PUMA at mRNA and protein levels were evaluated by SYBR Green quantitative PCR and Western blot, respectively.</p><p><b>RESULTS</b>Compared with HepG2/pcDNA3.1 cells (11.46% ± 0.69%) and HepG2 cells (12.5% ± 0.66%), the apoptosis rate of HepG2/HBx cells (2.37% ± 0.35%) was significantly reduced (F = 171.722, P < 0.01). The level of miR-192 was 49.1% ± 5.9% in HepG2 cells, which was dramatically down-regulated (F = 14.319, P = 0.019) as compared to the other two groups (HepG2/pcDNA3.1: 98.0% ± 8.9%; HepG2: 100%). Compared with HepG2 cells transfected with miR-NC (10.74% ± 1.15%), transfection of miR-192 into HepG2 cells led to increased apoptosis (15.74% ± 1.17%) (F = 18.415, P = 0.013) and higher p53 and PUMA expressions at mRNA (p53: 1.68 ± 0.12 vs 0.90 ± 0.09, F = 43.115, P = 0.003, PUMA: 1.66 ± 0.10 vs 0.98 ± 0.06, F = 22.541, P = 0.009) and protein (p53: 3.07 vs 1, PUMA: 2.13 vs 1) levels.</p><p><b>CONCLUSION</b>HBx could inhibit apoptosis of HepG2 cells through down-regulation of miR-192 which induces apoptosis of HepG2 cells.</p>