Influence of cytotoxic T lymphocyte-associated antigen 4 polymorphisms on the outcomes of hepatitis B virus infection.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) regulates T-cell activation and Th1/Th2 cytokine production and is involved in the immune response against Hepatitis B virus (HBV) infection. To detect the association of the CTLA-4 gene polymorphisms with susceptibility to HBV infection a hospital-based case-control study was conducted. A total of 1,119 unrelated individuals were recruited. The CTLA-4 variants rs5742909, rs231775 and rs3087243 were genotyped via the TaqMan method in this cohort. A comparison with a chronic active hepatitis B group revealed that the SNP rs231775 exhibited significant susceptibility to HBV progression, with the highest odds ratio (OR) reaching 1.659 and P=0.009-0.049. Although an HBV clearance group was used as a control, results of the present study demonstrated an association of rs5742909 with viral persistence [OR=1.694, 95% confidence intervals (CI)=1.124-2.553 and P=0.012]. Subsequent analyses revealed risk haplotypes (C-A-A and T-A-G, for which the highest OR reached 1.865) compared with the protective haplotype C-G-G. Therefore, SNPs in the CTLA-4 gene may be associated with HBV progression and viral persistence which is consistent with its emerging role in the T regulatory cells in the pathogenesis of disease.

[WISP-1: a novel mediator of hepatitis B virus-related hepatocellular carcinoma].

OBJECTIVE: To explore the effect of hepatitis B virus (HBV) X protein (HBX) on expression of the host gene Wnt induced secreted protein-1 (WISP-1) that is related to the pathogenic process of hepatocellular carcinoma (HCC). METHODS: Tumor and paratumor tissues were collected from HCC patients, and normal liver tissues were collected from healthy controls. Immunohistochemistry was used to evaluate the in vivo presence and expression levels of HBX and WISP-1 in the three tissue types. HepG2 cells stably transfected with pc-DNA3.1(+)-HBX or with pc-DNA3.1(+) only (G0, control) were generated and used to examine in vitro the HBX-induced changes in WISP-1 expression at the mRNA and protein levels by reverse transcription polymerase chain reaction and western blotting, respectively. RESULTS: The HCC tissues showed significantly higher rates of positivity for WISP-1 expression than the non-tumor controls (76.6% vs. paratumor: 23.4% or normal tissues: 0%, x2= 35.967, P less than 0.01). HBX increased WISP-1 expression in HepG2 cells at both the mRNA (1170.33 +/- 41.26 vs. G0: 265.34 +/- 27.47, t = 31.63, P less than 0.01) and protein (240.33 +/- 11.37 vs. G0: 40.33 +/- 7.09, F = 600.57, P less than 0.01) levels. CONCLUSION: HBV may up-regulate expression of the host gene WISP-1 through its X protein and thus promote the development of HCC.

[The effect of hepatitis B virus X protein on the expression of CtIP in HepG2 Cells].

To investigate the effect of hepatitis B virus X protein(HBx) on CtBP-interacting protein(CtIP) which is an important repair factor of DNA double strand break damage in HepG2 cells induced by bleomycin. A HBx stably expressing HepG2 cell line and a control HepG2 cell line with empty vector transfected were established. After the double strand break (DSB) damage occurred, the mRNA and protein levels of CtIP were detected by Real-time PCR and Western blot assay respectively, cell cycle profiles and apoptotic cell death were determined by a flow cytometry, and the position of CtIP in cells was observed by confocal laser scanning microscopy. It showed that HepG2 cells transfected with hepatitis B virus X gene could stably express HBx protein. After being induced by bleomycin, the percentage of apoptotic cell was 16.90%+/-0.89% in HBx stably expressing HepG2 cell line and 15.30%+/-0.86% in control cell line, respectively (q = 2.074, P is more than to 0.05). While the percentage of death cell was 8.71%+/-0.74% in HBx stably expressing HepG2 cell line and 4.90%+/-0.46% in control cell line, respectively (q = 7.126, P is less than to 0.01). The two cell lines manifested the increase of G2/M arrest and significant difference existed between the two cell lines. HBx down regulated the expression levels of CtIP and its mRNA. The CtIP level was 0.66+/-0.04 in HepG2-HBx cell and 0.73+/-0.05 in HepG2-vec cell, respectively (t = 2.314, P is less than to 0.05). The relative mRNA level was 1.00+/-0.06 in HepG2-HBx cell and 1.23+/-0.08 in HepG2-vec cell, respectively (t = 2. 732, P is less than to 0.05). We also found that CtIP was concentrated in the cell nucleus. The research suggests that HBx may affect DNA-repair pathways by disrupting the expression of CtIP.

MiR-192 inhibits nucleotide excision repair by targeting ERCC3 and ERCC4 in HepG2.2.15 cells.

Deficient DNA repair capacity is associated with genetic lesions accumulation and susceptibility to carcinogenesis. MicroRNAs (miRNAs) are small non-coding RNAs that regulate various cellular pathways including DNA repair. Here we hypothesized that the existence of HBV products may interfere with cellular nucleotide excision repair (NER) through microRNA-mediated gene regulation. We found that NER was impaired in HepG2.2.15 cells, a stable HBV-expressing cell line, compared with its parental cell line HepG2. Altered miRNA expression profile, in particular the significant upregulation of miR-192, was observed in HepG2.2.15 cells. Additionally, ERCC3 and ERCC4, two key factors implicated in NER, were identified as targets of miR-192 and over-expressing miR-192 significantly inhibited cellular NER. These results indicated that persistent HBV infection might trigger NER impairment in part through upregulation of miR-192, which suppressed the levels of ERCC3 and ERCC4. It provides new insight into the effect of chronic HBV infection on NER and genetic instability in cancer.

[HBx gene down-regulates miR-192 expression and inhibits apoptosis of human hepatoma cell line HepG2].

OBJECTIVE: To explore the mechanism by which HBV X gene(HBx) inhibits apoptosis of human hepatoma cell line HepG2 in terms of miRNA. METHODS: Three cell lines were prepared: HepG2 cells stably transfected with HBx (HepG2/HBx), HepG2 cells stably transfected with pcDNA3.1 (HepG2/pcDNA3.1) and HepG2 cells. Flow cytometry was adopted to measure the apoptosis of these three cells and Taqman fluorescence quantitative PCR was used to examine miR-192 expression. After HepG2 cells was transfected with miR-192, the apoptosis was analyzed by flow cytometry and the expressions of p53 and PUMA at mRNA and protein levels were evaluated by SYBR Green quantitative PCR and Western blot, respectively. RESULTS: Compared with HepG2/pcDNA3.1 cells (11.46% ± 0.69%) and HepG2 cells (12.5% ± 0.66%), the apoptosis rate of HepG2/HBx cells (2.37% ± 0.35%) was significantly reduced (F = 171.722, P < 0.01). The level of miR-192 was 49.1% ± 5.9% in HepG2 cells, which was dramatically down-regulated (F = 14.319, P = 0.019) as compared to the other two groups (HepG2/pcDNA3.1: 98.0% ± 8.9%; HepG2: 100%). Compared with HepG2 cells transfected with miR-NC (10.74% ± 1.15%), transfection of miR-192 into HepG2 cells led to increased apoptosis (15.74% ± 1.17%) (F = 18.415, P = 0.013) and higher p53 and PUMA expressions at mRNA (p53: 1.68 ± 0.12 vs 0.90 ± 0.09, F = 43.115, P = 0.003, PUMA: 1.66 ± 0.10 vs 0.98 ± 0.06, F = 22.541, P = 0.009) and protein (p53: 3.07 vs 1, PUMA: 2.13 vs 1) levels. CONCLUSION: HBx could inhibit apoptosis of HepG2 cells through down-regulation of miR-192 which induces apoptosis of HepG2 cells.

Persistent effect of IFNAR-1 genetic polymorphism on the long-term pathogenesis of chronic HBV infection.

Genetic polymorphism of IFNAR-1 plays a large role in determining the clearance or chronicity after hepatitis B virus (HBV) exposure. However, it is not clear whether type I interferon receptor-1 (IFNAR-1) variations continuously exert their effects to influence the final outcomes following HBV chronicity, including acute-on-chronic hepatitis B liver failure (ACLF-HBV), chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). Here we report that these four potential outcomes of chronic HBV infection are strongly associated with IFNAR-1 polymorphisms through a hospital-based case-control study of 663 cases. ACLF-HBV and HCC were each compared with CHB+LC. In comparison with the G/G genotype, the C/G and C/C genotypes at both single-nucleotide polymorphism (SNP) sites (rs1012335 and rs2257167) showed significant susceptibility to ACLF-HBV (the highest odds ratio [OR] reached 2.374; 95% CI = 1.488, 3.788; p < 0.001 for the C/G genotype at rs2257167), as well as HCC (OR = 2.475; 95% CI = 1.435, 4.426; p < 0.001 for the C/C genotype at rs1012335). The C allele at both loci was a susceptibility allele for ACLF-HBV and HCC, with the highest ORs reaching 1.653 (95% CI = 1.233, 2.216; p < 0.01 at rs1012335) in the ACLF-HBV group, and 1.659 (95% CI = 1.274, 2.159; p < 0.01 at rs1012335) in the HCC group. A strongly linked disequilibrium was also found within these two alleles (p < 0.001). Our research indicates that genetic polymorphisms of IFNAR-1 not only contribute to the determination of clearance or chronicity in the early stages of HBV exposure, but they also persistently influence pathogenesis over the long-term process of chronic HBV infection.