ABSTRACT
Objective:To explore the effects of hypoxia-inducible factor 2α genes on under hypoxia on proliferation,apoptosis, cell cycle distribution and migration of invasiveness of human hepatocellular carcinoma cell HepG2.Methods: Human hepatoma cell line HepG2 induced by cobalt chloride (CoCl2) was selected as the research object,construction of siRNA specific carrier HIF-2α, transfection of HepG2 cells under hypoxia.Real-time PCR,Western blot method in the detection of before and after transfection in each group of HIF-2α mRNA and protein expression;MTT method to detect the proliferation of HepG2 cells before and after transfection;apoptosis rate and distribution of cell cycle of HepG2 cells before and after transfection were detected by flow cytometry;Transwell test was used to detect the invasion and migration ability of HepG2 cells before and after transfection.Results: Under hypoxia,significant increased HIF-2α expression in hepatocellular carcinoma HepG2 cells.Specific transfection of HIF-2α siRNA in HepG2 cells after HIF-2α mRNA and protein expression levels were significantly inhibit cell proliferation decreased,apoptosis rate increased in the ratio of G0/G1 phase cells increased synthesis phase (S) and late (G2/M) synthesis cell ratio decreased,which in vitro invasion and migration of cells was inhibited.Conclusion:Expression of HIF-2α increases in hepatocellular carcinoma HepG2 cells under hypoxia. Specific siRNA can be cut by HIF-2α gene expression in HepG2 cells under hypoxia,to inhibit HepG2 cell proliferation,invasion, migration,and change the distribution of cell cycle and induce its apoptosis.
ABSTRACT
Objective To study the influence of targeted silencing of DEK on the proliferation and cell cycle of human hepatoma cell lines.Methods The human hepatoma cells line HepG2 were routinely cuhured and the cells were divided into blank control group,siRNA control group and DEK siRNA group when the cells grew to 90％ tusion.The cells in blank control group were cultured normally without any treatment;the cells in siRNA control group and DEK siRNA group were transfected with siRNA expression vector and DEK siRNA expression vector mediated by LipofectamineTM2000 liposomes,respectively.The expression of DEK mRNA in HepG2 cells was detected by real-time polymerase chain reaction;the expression of DEK and CyclinD1 protein in HepG2 cells was detected by Western blot;the proliferation of HepG2 cells was detected by methyl thiazolyl tetrazolium method,and the cell cycle was observed by flow cytometry.Results The expression of DEK mRNA in the blank control group,siRNA control group and DEK siRNA group was 0.826 ±0.052,0.776 ±0.051 and 0.420 ±0.050 respectively;the expression of DEK protein in the blank control group,siRNA control group and DEK siRNA group was 0.691 ± 0.073,0.726±0.061 and 0.311 ±0.038 respectively;the expression of CyclinDl protein in the blank control group,siRNA cuntrol group and DEK siRNA group was 0.712 ± 0.069,0.780 ± 0.074 and 0.434 ± 0.039 respectively.The expressions of DEK mRNA,DEK protein and CyclinD1 protein in DEK siRNA group were significantly lower than those in the blank control group and siRNA control group (P ＜ 0.05);there was no statistic difference in the expression of DEK mRNA,DEK protein and CyclinD1 protein between the blank control group and siRNA control group(P ＜0.05).The proliferation ability of HepG2 cells in DEK siRNA group after transfection of 24,48,72,96,120 h was significantly lower than that in the blank control group and siRNA control group(P ＜0.05);there was no statistic difference in the proliferation ability of HepG2 cells between the blank control group and siRNA control group at each time point(P ＜ 0.05).The proportion of G0 + G1 phase cells in DEK siRNA group was significantly higher than that in the blank control group and siRNA control group(P ＜ 0.05);the proportions of S phase and G2 + M phase cells in DEK siRNA group were significantly lower than those in the blank control group and siRNA control group(P ＜ 0.05);there was no statistic difference in the proportion of G0 + G1 phase,S phase and G2 + M phase cells between the blank control group and siRNA control group (P ＜ 0.05).The result of Pearson correlation analysis showed that the expression of CyclinD1 protein was positively correlated with the expression of DEK mRNA and protein(r =0.909,0.899;P ＜ 0.05).Conclusion DEK siRNA can inhibit the proliferation of HepG2 cells,and change the cell cycle distribution through down regulating the expression of DEK gene in HepG2 cells.This process may be related to the down regulation of the expression of CyclinD1.
ABSTRACT
MTT assay was used in this study to investigate the inhibitory effect of danshensu on the activity of 2.2.15 cells among human hepatoma cell line (HepG2); indirect fluorescence labeling method was used to measure the changes of reactive oxygen levels in the cells; ELISA method was used to determine hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels in cellular supernatants; HBV DNA level was measured with fluorogenic quantitative PCR method. The inhibitory effect of danshensu on HBV RT(hepatitis B virus reverse transcriptase) was studied by using enzyme inhibition dynamics, and the effect of danshensu on secondary structure of HBV reverse transcriptase was monitored by using circular dichroism. The results showed that danshensu had a good inhibitory effect on the growth of HepG2.2.15 cells, with a half inhibitory concentration (IC₅₀) of (15.35±2.43) μmol•L⁻¹; danshensu could significantly inhibit HBsAg and HBeAg expressions, and showed an inhibitory effect on HBV DNA replication. In addition, danshensu was an effective inhibitor for HBV reverse transcriptase [IC₅₀ (21.32±2.43) μmol•L⁻¹]. The fluorescence labeling results showed that the reactive oxygen levels in the cells were increased with the increase of danshensu concentration. Circular dichroism analysis showed that danshensu could induce partial change of conformation of HBV reverse transcriptase and gradually increased α-helical content. These results indicated that danshensu could make the structure of the enzyme become closer by binding to HBV reverse transcriptase, which was not conducive to the formation of the active center, so it could finally decrease the activity of HBV reverse transcriptase. Such decrease in enzyme activity would directly affect the HBV DNA replication, and combined with the decrease of the antigen levels, the effect of danshensu on HBV was increased.
ABSTRACT
<p><b>OBJECTIVE</b>To study the effect of oxymatrine on serum cytokines and hepatic fibrotic indexes in patients with chronic hepatitis B (CHB).</p><p><b>METHODS</b>Eighty-two CHB patients were randomly assigned to the control group treated with Western routine therapy and the treated group treated by Western routine therapy plus oxymatrine. The treatment course was 24 weeks. Another group with 20 healthy subjects was set as the normal control group. The serum levels of transforming growth factor-beta1 (TGF-beta1), interleukin-6 (IL-6), hyaline acid (HA), collagen type IV (C-IV) and laminin (LN) were measured by ELISA and RIA before and after treatment.</p><p><b>RESULTS</b>The serum levels of TGF-beta1, IL-6, HA, C-IV and LN in CHB patients were significantly higher than those in the normal control group (P < 0.01). After 24-week treatment with oxymatrine, these abnormal indexes in the treated group were significantly lowered, as compared with those before treatment and in the control group, the differences were significant (all P < 0.01).</p><p><b>CONCLUSION</b>Oxymatrine could lower the levels of cytokines, including TGF-beta1, IL-6, etc. in patients with CHB, which might be one of the mechanisms of its action in reversing liver fibrosis.</p>