Effect of HBx gene RNA interference combined with chemotherapy on hepatocellular carcinoma cells / 中南大学学报(医学版)

OBJECTIVE@#To determine the influence of HBx gene RNA interference combined with chemotherapy on stable hepatocellular carcinoma cells growth and its apoptosis mechanism.@*METHODS@#Stable hepatocellular carcinoma cells transfected by shRNA aiming at HBx together with independent control series (MHCC97-H,HK3, and 21543) were identified. The extent of HBx gene by RNA interference was detected by RT-PCR. The influence of cell growth through RNA interference was observed with cell counting kit-8 (CCK8), the diversity of cell cycle by flow cytometry and cell apoptosis were detected by TUNEL apoptosis detection kit.@*RESULTS@#RT-PCR demonstrated that the HBx mRNA level of 21,543 cell down regulation was 91%. The HBx mRNA level of HK3 cells was not different from MHCC97-H cell. The growth of 21,543 cells was obviously slower than MHCC97-H cells and HK3 cells, with no significant difference. The cell cycle of 21,543 cells showed that hepatocellular carcinoma cells through RNA interference targeting at HBx delayed in go to S stage, and the proliferation activity degraded obviously. The 3 kinds of cells adding different concentrations of flurouracil and cisplatin grew slowlier than the origin cells. The growth inhibition was dependent on the concentration of drug with growth inhibition of 21,543 cells the most obvious.That of the 3 kinds of cells adding alpha-interferon was not obvious.Flurouracil induced apoptosis in all cells. Apoptosis in 21,543 cells was the most obvious.@*CONCLUSION@#RNA interference targeting at HBx can suppress the growth of hepatocellular carcinoma cells. Hepatocellular carcinoma cells through RNA interference targeting at HBx can intensify chemo-sensitivity. Combination of RNA interference targeting at HBx with chemotherapeutics can induce apoptosis in more hepatocellular carcinoma cells and cell proliferation steps down accordingly.

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>

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.