Downregulation of xIAP expression by small interfering RNA inhibits cellular viability and increases chemosensitivity to methotrexate in human hepatoma cell line HepG2.

The aim of this study was to investigate whether downregulating the expression of xIAP by RNAi (RNA interference) technology can induce the apoptosis of HepG2 cells, inhibit cellular viability and increase chemosensitivity of cancer cells. HepG2 cells were transfected with U6 promoter plasmids coding for short interfering RNAs (siRNAs) targeting xIAP. RT-PCR and western blot analysis were used to assess the mRNA and protein levels of xIAP expression. T he suppression efficiency o f xIAPby RNAi was evaluated using the MTT assay for cellular viability and Annexin V-PI binding assay for apoptosis. These results showed that siRNAs reduced cellular viability and increased cellular apoptosis. Moreover, downregulation of xIAP expression enhanced the chemosensitivity of HepG2 cells to methotrexate. These results suggest that the downregulation of xIAP by RNAi could potentially be used as a therapeutic strategy for human hepatocellular carcinoma.

[Molecular mechanism of HCV NS5A on p53's inhibition of AFP expression in hepatocellular carcinoma cells].

OBJECTIVE: To explore hepatitis C virus (HCV) non-structural protein 5A (NS5A)'s influence on inhibition of AFP expression executed by p53 protein and its possible molecular mechanism. METHODS: Plasmid transfection and MEIA were employed to observe p53's inhibitive effect on AFP expression of Huh7 cells and the HCV NS5A's influence on p53 function. Western blot was employed to find out if HCV NS5A affects p53 protein expression and GST pull down assay was applied to examine the interaction between HCV NS5A and p53. RESULTS: The AFP concentration in the supernatant of the culture of the Huh7 cells transfected with pRc/CMV was (14322+/-2412) ng/ml, and that of the Huh7 cells transfected with pCNS5A was (13843+/-3218) ng/ml; no significant difference existed between these two groups (t = 1.42, P > 0.05). After transfection with pC53-NS3, the AFP level was decreased to (10 241+/-1326) ng/ml, and in comparison to the above two groups it had a statistically significant difference (t values were 2.41 and 2.38, P < 0.05). When co-transfected with pCNS5A and pC53-NS3, the AFP expression (14582+/-1238) ng/ml returned to the level of pRc/CMV transfected, and there was a remarkably significant difference between this and that of the pC53-NS3 transfected cells (t = 3.12, P < 0.01). HCV NS5A had no function on the p53 protein expression with Western blot experiment. In the GST pull down assay, an HCV NS5A protein band was found after GST-p53 was added, but not detected with GST only. CONCLUSION: We found that p53 has an inhibitive function on the AFP expression in Huh7 cells and HCV NS5A minimized this p53 function. HCV NS5A did not affect p53 protein expression, but was able to form a complex with p53, by which HCV NS5A inactivated this p53 function.

Transfection of apoptosis related gene Fas ligand in human hepatocellular carcinoma cells and its significance in apoptosis.

AIM: To evaluate the expression of apoptosis related gene Fas ligand (FasL) in human hepatocellular carcinoma (HCC) cells HepG2 and its significance in apoptosis. METHODS: Levels of soluble Fas ligand (sFasL) in a group of patients with hepatitis B virus (HBV)-induced chronic hepatitis, HBV-positive liver cirrhosis and HCC were evaluated. In a further study, the recombinant eukaryotic expression plasmid pcDNA3.1hisB-FasL was transfected into HCC cells HepG2 by lipofection, and then soluble FasL was examined in the supernatant of culture cells by EIA, FasL expression in HepG2 cells was detected by immunohistochemistry. After being stained by annexin V and propidium iodine, cells were passed through a flow cytometer and examined by a fluorescence microscope and a laser scanning microscope. RESULTS: The sFasL levels were significantly lower in patients with HCC when compared to the patients with hepatitis or liver cirrhosis. In comparison with untransfected cells, the soluble FasL could be detected in the supernatant of transfected cells. FasL was expressed on the membranes and cytoplasm of transfected cells. The apoptotic cell rate was 36.30% in transfected cells, and was 11.53% in untransfected cells. Moreover, the different stage of apoptotic cells could be distinguished by annexin V and propidium iodine staining. CONCLUSION: Fas ligand is an apoptotic pathway of HCC cells.

HCV NS5A abrogates p53 protein function by interfering with p53-DNA binding.

AIM: To evaluate the inhibition effect of HCV NS5A on p53 transactivation on p21 promoter and explore its possible mechanism for influencing p53 function. METHODS: p53 function of transactivation on p21 promoter was studied with a luciferase reporter system in which the luciferase gene is driven by p21 promoter, and the p53-DNA binding ability was observed with the use of electrophoretic mobility-shift assay (EMSA). Lipofectin mediated p53 or HCV NS5A expression vectors were used to transfect hepatoma cell lines to observe whether HCV NS5A could abrogate the binding ability of p53 to its specific DNA sequence and p53 transactivation on p21 promoter. Western blot experiment was used for detection of HCV NS5A and p53 proteins expression. RESULTS: Relative luciferase activity driven by p21 promoter increased significantly in the presence of endogenous p53 protein. Compared to the control group, exogenous p53 protein also stimulated p21 promoter driven luciferase gene expression in a dose-dependent way. HCV NS5A protein gradually inhibited both endogenous and exogenous p53 transactivation on p21 promoter with increase of the dose of HCV NS5A expression plasmid. By the experiment of EMSA, we could find p53 binding to its specific DNA sequence and, when co-transfected with increased dose of HCV NS5A expression vector, the p53 binding affinity to its DNA gradually decreased and finally disappeared. Between the Huh 7 cells transfected with p53 expression vector alone or co-transfected with HCV NS5A expression vector, there was no difference in the p53 protein expression. CONCLUSION: HCV NS5A inhibits p53 transactivation on p21 promoter through abrogating p53 binding affinity to its specific DNA sequence. It does not affect p53 protein expression.

[Cloning and sequence of human hepatocyte DNA synthetic stimulated factor cDNA].

OBJECTIVE: To clarify the nature of human hepatocyte DNA synthetic stimulated factor (HDSSF) and to lay a foundation for its gene cloning and expression. METHODS: We amplified the target gene fragment by degenerating PCR, labelled the fragment as a probe to screen human fetal liver cDNA library, and then analyzed the HDSSF gene sequence. RESULTS: A 600 base pair fragment was obtained and labelled successfully with DIG. Human HDSSF sequence was determined by screening the library and gene sequencing. Its cDNA chain was composed of 748 base pairs including 594 base pair open read-code frame and 5' and 3' terminal noncode district sequence. CONCLUSION: By screening cDNA library, the HDSSF gene cloning has been gained successfully. HDSSF should be a new hepatocyte growth substance which consists of 198 amino acid residues.

[Quantitative detection of hepatitis B virus by fluorescence quantitative PCR and its clinical value].

OBJECTIVE: To study the relationship between the serum contents of HBV DNA copies and the HBV serum index of immunology and the degree of hepatocyte injury in patients with HBV hepatitis. METHODS: The contents of HBV DNA copies were detected by fluorescence quantitative PCR (FQ-PCR), and the HBV serum index of immunology by ELISA. RESULTS: The contents of HBV DNA copies had a significant difference between HBeAg (+)/anti-HBe (-) group (42 patients) and HBeAg (-)/anti-HBe (+) group (57 patients) or HBeAg (-)/anti-HBe (-) group(11 patients) (P < 0.05), while there was no significant difference between HBeAg (-)/anti-HBe (+) group and HBeAg (-)/anti-HBe (-) group(P > 0.05). The contents of HBV DNA copies had no correlation with the severe degree of hepatocyte injury(P > 0.05). CONCLUSION: Anti-HBe is not the terminal sign of HBV replication. The severe degree of hepatocyte injury has not a direct correlation with the contents of HBV DNA copies.

[Regulation mechanism of HCV NS5A on p53 protein transactivity].

OBJECTIVE: To study the inhibition effect of HCV NS5A on p53 protein transactivity and its possible mechanism. METHODS: Luciferase reporter gene system was used for the study of p53 transactivity on p21 promoter and electrophorectic mobility-shift assay (EMSA) was applied to observe whether HCV NS5A could suppress the binding ability of p53 protein to its specific DNA sequence. RESULTS: Endogenous p53 protein could stimulate p21 promoter activity, and the relative luciferase activity increased significantly (3.49 x 10(5) vs 0.60 x 10(5), t = 5.92, P<0.01). Exogenous p53 protein also up-regulated p21 promoter driving luciferase expression, comparing to the control group (0.47 x 10(5)), the relative luciferase activity increased (5.63 x 10(5)) obviously (t = 10.12, P<0.01). HCV NS5A protein inhibited both endogenous and exogenous p53 transactivity on p21 promoter in a dose-dependent manner (F > or = 20.71, P<0.01). In the experiment of EMSA, p53 could bind to its specific DNA sequence, but when co-transfected with HCV NS5A expressing vector, the p53 binding affinity to its DNA decreased. CONCLUSION: HCV NS5A can inhibit p53 protein transactivity on p21 promoter through its inhibiting of p53 binding ability to the specific DNA sequence.

HCV replication in PBMC and its influence on interferon therapy.

AIM: To study hepatic virus C (HCV) RNA and HCV protein expression in peripheral blood mononuclear cells (PBMCs) of patients with HCV infection, and explore the relationship between the HCV RNA in the PBMCs and response to interferon (IFN) therapy. METHODS: Type-specific primers were designed and RT-nested PCR was used to detect the plus- and minus- strands of HCV RNA in PBMCs of 54 patients with HCV infection; Indirect immunofluorescence assay was applied to identify HCVNS5 protein expression in PBMCs; 6 month-, 3 MU-IFN regiment was administrated to observe the responses to IFN in 35 chronic hepatitis C patients with different HCV RNA status in PBMCs. RESULTS: HCV plus strand RNA was found in 10 of 19 (52.6 %) acute hepatitis C patients and 22 of 35 (62.9 %) chronic hepatitis C patients. HCV minus strand RNA was detected in 14 of 35 (40.0 %) chronic hepatitis C patients, but only one patient (5.3 %) with acute HCV infection was found to be minus HCV RNA positive. Though no HCV NS5 protein expression was found in the examined 10 cases of acute HCV infection, it was positive in 17 of 20 (85.0 %) chronic hepatitis C patients by indirect immunofluorescence assay. There are significant differences of positive rate of the minus-strand and HCVNS5 protein between acute and chronic hepatitis C groups (u=2.07, P<0.05 and u=4.43, P<0.01 respectively). The patients with minus-strand HCV RNA showed a significantly lower 6-month sustained response (SR-6) to IFN compared to those without minus-strand HCVRNA in PBMCs (biologically 14.3 % vs 42.8 %, chi(2)=4.12, P<0.05 and virologically 7.1 % vs 23.9 %, chi(2)=4.24, P<0.05). CONCLUSION: HCV is capable of infecting and replicating in PBMCs, and HCVNS5 protein was expressed in PBMCs. The patients with minus strand HCV RNA in PBMCs showed a significantly lower 6-month sustained response to IFN, suggesting that minus-strand HCV RNA in PBMCs may be one of the factors influencing response to IFN therapy.

[Construction of high efficient eukaryotic expression recombinant on human hepatocyte DNA synthetic stimulated factor].

OBJECTIVE: To construct the eukaryotic expression recombining vector on human The pGEM-hepatocyte DNA synthetic stimulated factor (hHDSSF) with gene cloning. METHODS: hHDSSF, a mid-recombining vector, was constructed by T-A cloning. After restriction endonuclease Not I digestion, the target fragment was subcloned into eukaryotic vector pcDNA3. 1hisB to construct the eukaryotic expression recombinant pcDNA3. 1hisB-HDSSF. RESULTS: The forward insert recombinant pcDNA3. 1hisB-HDSSF was screened and obtained with restriction endonuclease Kpn I digestion and it was detected by DNA sequence analysis. CONCLUSION: The eukaryotic expression recombinant pcDNA3. 1hisB-hHDSSF on hHDSSF is constructed successfully, which lays a foundation for building a stable eukaryotic expression cell strain and expressing hHDSSF.