Interleukin-32 expression in serum of patients with HBV-related liver failure and its significance / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the expression level of IL-32 in serum and its correlation with serum biochemical indices of liver function test and HBV DNA load in patients with HBV-related liver failure.</p><p><b>METHODS</b>Fifty-five patients with HBV-related liver failure (severe hepatitis group) and twenty normal cases (control group) were enrolled in the study. Total RNA in PBMCs was extracted by using TRIzol. IL-32 mRNA level was assayed by using Real-time PCR. IL-32 protein level in serum was detected by ELSIA method. The correlation between IL-32 and ALT, AST, TBIL, HBV DNA load was analyzed using pearson's correlation analysis, respectively.</p><p><b>RESULTS</b>Serum IL-32 expression level in severe hepatitis group was higher than that of control group. Moreover, the difference between them was statistically significant (P < 0.05). Serum IL-32 level was positively correlated with serum ALT, AST, TBIL, respectively (P < 0.05), but was not correlated with HBV DNA load (P > 0.05).</p><p><b>CONCLUSION</b>Serum IL-32 expression level was increased in patients with HBV-related liver failure and was associated with the severity of inflammation. We, therefore, believe that IL-32 might be involved in the pathogenesis of HBV-related liver failure.</p>

Hepatitis B virus X promotes HepG2 cell cycle progression and growth via downregulation expression of p16 protein / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effects and related mechanisms of hepatitis B virus X (HBx) protein on cell cycle and growth in hepatocellular carcinoma.</p><p><b>METHODS</b>A human hepatocyte HepG2 cell line stably expressing a green fluorescent protein (GFP)-tagged HBx (HepG2/GFP-HBx cells) was used for the experiment, and HepG2 parental and HepG2/GFP cells was used as the controls. Effect of HBx on cell growth was evaluated by the MTT cell proliferation assay and on cell cycle progression by flow cytometry analysis of cells with or without treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR; 5 pmol/L). Effect of HBx expression on promoter methylation status of the p16INK4A tumor-suppressor gene was detected by methylation-specific polymerase chain reaction and on p16 protein level was analyzed with western blotting.</p><p><b>RESULTS</b>The HepG2/GFP-HBx cells showed significantly higher cell proliferation at 72 hrs of culture (3.225+/-0.038 A490) than either control (HepG2: 2.012+/-0.022 A490, t = -46.86, P less than 0.001; HepG2/GFP: 2.038+/-0.029 A490, t = 42.51, P less than 0.001). The HepG2/GFP-HBx cells also showed significantly lower proportion of cells in the G0/G1 phase (16.45%+/-0.45%) than either control (HepG2: 44.81%+/-1.36%, t = -34.202, P less than 0.001; HepG2/GFP: 42.76%+/-1.58%, t = -28.88, P less than 0.001). However, 5-Aza-CdR treatment did lead to a significant amount of HepG2/GFP-HBx cells being arrested in the G0/G1 phase (33.25%+/-0.79%, t = 31.85, P less than 0.001). The p16INK4A promoter was methylated in the HepG2/GFP-HBx cells, and became demethylation after treatment with 5-Aza-CdR. However, no methylation of p16INK4A promoter was observed in both HepG2 and HepG2/GFP cells. The p16 protein level was significantly lower in the HepG2/GFP-HBx (vs. HepG2 and HepG2/GFP cells) and this level increased after treatment with 5-Aza-CdR.</p><p><b>CONCLUSION</b>HBx protein promotes hepatocellular carcinoma cell cycle progression and growth by shortening the G0/G1 phase, and the underlying mechanism may involve inducing p16INK4A promoter methylation and downregulating p16 protein expression.</p>

Small interfering RNA targeting to hepatitis B virus X gene and 5-aza-2'-deoxycytidineon inhibited growth of the subcutaneous implanted tumor of hepatocellular carcinoma in nude mice / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the anti-tumor effect of small interfering RNA targeting to HBV X gene (X-siRNA) and 5-aza-2'-deoxycytidine (5-aza-dC) on HBV-related hepatocellular carcinoma.</p><p><b>METHODS</b>X-siRNA and control siRNA were synthesized. HepG2/GFP-HBx cells were treated with X-siRNA, and the levels of HBV X mRNA were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Nude mice were inoculated with HepG2/GFP and HepG2/GFP-HBx cells subcutaneous respectively to establish implant models of hepatocellular carcinoma, and were treated with X-siRNA, 5-aza-dC alone or in combination, and tumor growth was observed. The methylation of p16 gene promoter was detected by methylation specific polymerase chain reaction (MSP).</p><p><b>RESULTS</b>RT-PCR showed the expression of HBV X mRNA in HepG2/GFP-HBx cells was inhibited markedly by X-siRNA. The nude mice experiment showed that the gross tumor volume was much bigger in HepG2/GFP-HBx group than that in HepG2/GFP group (P < 0.05). The growth of palpable tumors in X-siRNA or 5-aza-dC treatment group notably decreased (P < 0.05). MSP analysis showed that p16 gene methylation was observed in HepG2/ GFP-HBx-caused palpable tumors, while no methylation was detected in HepG2/GFP group. However, after treatment with X-siRNA or 5-aza-dC, p16 gene methylation reduced.</p><p><b>CONCLUSIONS</b>HBV X-siRNA and methylation inhibitor can inhibit the growth of hepatoma cells via reversing p16 methylation.</p>