Effects of HBV X gene and arsenic trioxide on the expression of p53 in cultured HepG2 cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Hepatitis B virus (HBV) X protein (HBx) and p53 could mutually down-regulate at transcriptional level and HBx could bind with p53 protein within its transactivation domain and inhibit the function of p53 protein. In recent years, effects of arsenic trioxide (As2O3) on the expression of p53 protein have been widely studied, while little is known about the activity of p53 protein. This study was undertaken to delineate the effect of HBV X gene and As2O3 on p53 protein expression (level and activity) in HepG2 cells by small hairpin RNA (shRNA)-mediated RNA interference (RNAi) technique.</p><p><b>METHODS</b>Cell line HepG2 and cells with stable expression of HBV X gene (HepG2-X) were treated with 2 micromol/L As2O3, with corresponding untreated cells serving as controls. Cell lysates and nuclear extracts were extracted. Total level and the relative activity of p53 protein were detected by modified enzyme-linked immunosorbent assay (ELISA). HBV X gene sequence-specific shRNA expression vector (pXi-1 and pXi-2) and sequence-unrelated control (pXi-3) were transfected into HepG2-X. Single cell clone with stable expression of shRNA was selected and exposed to propagating culture. The effect of As2O3 on p53 protein expression and activity was re-observed.</p><p><b>RESULTS</b>Total p53 protein level was up-regulated and its relative activity ratio was enhanced by As2O3 in HepG2 and HepG2-X cells. The total p53 protein level induced by As2O3 was up-regulated by HBV X gene expression, while its relative activity was significantly suppressed. The suppression was removed after HBV X gene expression was repressed by shRNA.</p><p><b>CONCLUSIONS</b>As2O3 up-regulates p53 protein expression and enhance its activity. HBV X up-regulates As2O3 induced-p53 protein expression while suppresses its activity.</p>

Study of effects of HBV X gene and As2O3 on expression and activity of p53 in HepG2 cells with shRNA / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To delineate the effects of HBV X gene and of As(2)O(3) on p53 expression and activity in HepG2 cells by shRNA-mediated RNA interference (RNAi).</p><p><b>METHODS</b>HepG2 cells and cells with stable expression of HBV X gene, HepG2-X, were treated with 2 micromol/L As(2)O(3), and the corresponding untreated cells were used as controls. Cell and nuclear lysates were extracted. Total level and the relative activity absorbance of p53 were detected by modified ELISA. HBV X gene sequence-specific shRNA expression vectors, Xi-S1 and Xi-S2, and sequence-unrelated control Xi-S3 were transfected into HepG2-X. The effect of As(2)O(3) on p53 expression and activity were retested.</p><p><b>RESULTS</b>Total p53 level was up-regulated and its relative activity ratio was enhanced by As(2)O(3) in HepG2 and HepG2-X cells. The total p53 level induced by As(2)O(3) was further up-regulated by HBX expression, while its relative activity was significantly suppressed. The suppression was removed after HBX expression was suppressed by shRNA.</p><p><b>CONCLUSION</b>As(2)O(3) could up-regulate p53 expression and enhance its activity. shRNA-mediated RNA interference is conveniently being used in studies on the effect of HBV X gene expression on p53 expression and activity. HBV X expression could up-regulate p53 gene expression level induced by As(2)O(3), while it suppressed the activity of p53.</p>

Serum HBsAg concentration and HBV replication level in hepatitis B patients with positive serum HBsAg and HBeAg / 中南大学学报(医学版)

OBJECTIVE@#To analyze the relationship between serum HBsAg concentration and HBV replication level in hepatitis B patients with positive serum HBsAg and HBeAg, and to explore the possibility of using serum HBsAg concentration as a marker of HBV replication level in hepatitis B patients with positive serum HBeAg.@*METHODS@#HBV DNA level and serum HBeAg, HBsAg concentration of 296 patients with positive serum HBsAg and HBeAg were quantitatively detected by real-time fluorescence quantitative PCR (FQ-PCR) and time-resolved fluoroimmunoassay (TRIFA) respectively. HBsAg concentrations were compared among patients with different HBV DNA levels, and HBV DNA levels were compared among patients with different HBsAg concentrations. The correlation between serum HBsAg concentration and DNA replication level were analyzed. The positive, negative predictive values and coincidence rates were speculated by various HBsAg concentrations.@*RESULTS@#If HBV DNA positive was defined as HBV DNA levels no less than 10(5) copy/mL, then 228(77.03%) patients were classified as HBV DNA positive. HBsAg concentration was positively correlated with HBV DNA replication level, but among groups with various DNA replication levels, HBsAg concentration showed no significant statistical difference (P>0.05). If the patients were divided into 2 groups, HBsAg concentration (180 microg/L) was served as the cutoff level, the DNA positive rate of the group with HBsAg concentration no less than 180 microg/L was significantly higher than that with HBsAg concentration less than 180 microg/L (chi(2)=3.998, P<0.05). DNA positive rates and average DNA levels showed no significant statistical differences between the 2 groups, if HBsAg concentrations other than 180 microg/L were used as the cutoff level. Positive predictive values, negative predictive values and the coincidence rates speculated by various HBsAg concentrations as cutoff values did not show any significant statistical difference in estimating HBV replication levels.@*CONCLUSION@#To some extent, serum HBsAg concentration is related to HBV DNA replication level in hepatitis B patients with positive serum HBsAg and HBeAg, but it is not feasible to use HBsAg concentration to monitor their HBV replication levels.

Construction of two hepatocellular carcinoma cell models for the expression of HBV X gene with different selection characteristics / 中南大学学报(医学版)

OBJECTIVE@#To construct 2 hepatocellular carcinoma (HCC) cell models for the expression of HBV X gene with different selection characteristics.@*METHODS@#HepG2 HCC cells were infected with eukaryotic expression vectors with HBV X gene, pCEP4-X, and pcDNA3. 1 (+)-X. Single cell clone was selected by hygromycin and neomycin. After propagating culture for certain periods, the HBV X gene expression was identified by PCR, RT-PCR, and Western blot.@*RESULTS@#Single HCC cell clone with HBV X gene transferred resistant to hygromycin and neomycin was selectively cultured, and the cells could be propagated for certain periods. PCR, RT-PCR, and Western blot identified the expression of HBV X gene.@*CONCLUSION@#Two HCC cell models for the expression of HBV X gene with different selection characteristics have been successfully constructed.