Lnc-HUR1 is an HBV-related long non-coding RNA, which can promote the proliferation of hepatoma cells and the occurrence and development of liver cancer. In this study we explored the effect of lnc-HUR1 on the apoptosis of hepatocellular carcinoma cells by taking the approach of immunoblotting, quantitative real time PCR, luciferase reporter assay, chromatin immunoprecipitation (ChIP) and flow cytometry. We found that overexpression of lnc-HUR1 significantly reduced the activity of caspase3/7 and the cleavage of PARP-1, while knocking down of lnc-HUR1 significantly increased the activity of caspase3/7 and promoted the cleavage of PARP-1 in HepG2 cells treated with TGF-β, pentafluorouracil or staurosporine. Consistently, the data from Annexin-V/PI staining showed that overexpression of lnc-HUR1 inhibited apoptosis, while knockdown of lnc-HUR1 promoted apoptosis. Moreover, overexpression of lnc-HUR1 up-regulated the apoptosis inhibitor Bcl-2 and down-regulated the pro-apoptotic factor BAX at both RNA and protein levels. In the CCL4-induced acute liver injury mice model, the expression of Bcl-2 in the liver tissue of lnc-HUR1 transgenic mice was higher than that of the control mice. The data from ChIP assay indicated that lnc-HUR1 reduced the enrichment of p53 on Bcl-2 and BAX promoters. All these results indicated that lnc-HUR1 inhibited the apoptosis by promoting the expression of apoptosis inhibitor Bcl-2 and inhibiting the expression of apoptosis promoting factor BAX. Further studies showed that lnc-HUR1 regulated the transcription of Bcl-2 and BAX in HCT116 cells, but had no effect on the expression of Bcl-2 and BAX in HCT116 p53^-/- cells, indicating that lnc-HUR1 regulates the transcription of Bcl-2 and BAX dependent upon the activity of p53. In conclusion, HBV upregulated lnc-HUR1 can inhibit the apoptosis of hepatoma cells. Lnc-HUR1 inhibits apoptosis by inhibiting the transcriptional activity of p53. These results suggest that lnc-HUR1 plays an important role in the occurrence and development of HBV-related hepatocellular carcinoma.
Animals ; Annexins/pharmacology* ; Apoptosis ; Carcinoma, Hepatocellular/genetics* ; Cell Proliferation ; Hep G2 Cells ; Hepatitis B virus/metabolism* ; Humans ; Liver Neoplasms/genetics* ; Mice ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology* ; Proto-Oncogene Proteins c-bcl-2/pharmacology* ; RNA, Long Noncoding/metabolism* ; Staurosporine/pharmacology* ; Transforming Growth Factor beta/pharmacology* ; Tumor Suppressor Protein p53/pharmacology* ; bcl-2-Associated X Protein/pharmacology*

To investigate whether transcription of hepatitis B virus (HBV) gene occurs in human sperm, total RNA was extracted from sperm of patients with chronic HBV infection (test-1), from donor sperm transfected with a plasmid containing the full-length HBV genome (test-2), and from nontransfected donor sperm (control), used as the template for reverse transcription-polymerase chain reaction (RT-PCR). Positive bands for HBV DNA were observed in the test groups but not in the control. Next, to identify the role of host genes in regulating viral gene transcription in sperm, total RNA was extracted from 2-cell embryos derived from hamster oocytes fertilized in vitro by HBV-transfected (test) or nontransfected (control) human sperm and successively subjected to SMART-PCR, suppression subtractive hybridization, T/A cloning, bacterial amplification, microarray hybridization, sequencing and the Basic Local Alignment Search Tool (BLAST) search to isolate differentially expressed genes. Twenty-nine sequences showing significant identity to five human gene families were identified, with chorionic somatomammotropin hormone 2 (CSH2), eukaryotic translation initiation factor 4 gamma 2 (EIF4G2), pterin-4 alpha-carbinolamine dehydratase 2 (PCBD2), pregnancy-specific beta-1-glycoprotein 4 (PSG4) and titin (TTN) selected to represent target genes. Using real-time quantitative RT-PCR (qRT-PCR), when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNA interference, transcriptional levels of HBV s and x genes significantly decreased (or increased) (P < 0.05). Silencing of a control gene in sperm did not significantly change transcription of HBV s and x genes (P > 0.05). This study provides the first experimental evidence that transcription of HBV genes occurs in human sperm and is regulated by host genes.
Animals ; Connectin/genetics* ; Cricetinae ; Eukaryotic Initiation Factor-4G/genetics* ; Gene Expression Regulation/genetics* ; Gene Silencing ; Growth Hormone/genetics* ; Hepatitis B Surface Antigens/genetics* ; Hepatitis B virus/genetics* ; Hepatitis B, Chronic/virology* ; Humans ; Hydro-Lyases/metabolism* ; Male ; Pregnancy-Specific beta 1-Glycoproteins/genetics* ; RNA, Viral/analysis* ; Spermatozoa/virology* ; Trans-Activators/genetics* ; Transcription, Genetic ; Transfection ; Viral Regulatory and Accessory Proteins

BACKGROUND/AIMS: The clinical outcome of patients with a partial virological response (PVR) to entecavir (ETV), in particular nucloes(t)ide analogue (NA)-experienced patients, has not been thoroughly investigated. The aim of the present study was to assess long-term outcomes in NA-naive and NA-experienced chronic hepatitis B patients with a PVR to ETV. METHODS: Chronic hepatitis B patients treated with ETV (0.5 mg/day) for at least 1 year were enrolled retrospectively. PVR was defined as a decrease in hepatitis B virus (HBV) DNA titer of more than 2 log10 IU/mL, yet with residual serum HBV DNA, as determined by real time-polymerase chain reaction, at week 48 of ETV therapy. RESULTS: A total of 202 patients (127 NA-naive and 75 NA-experienced, male 70.8%, antigen positive 53.2%, baseline serum HBV DNA 6.2 +/- 1.5 log10 IU/mL) were analyzed. Twenty-eight patients demonstrated a PVR. The PVR was associated with a high serum HBV DNA titer at baseline and at week 24. Virological response (< 60 IU/mL) was achieved in 46.2%, 61.5%, 77.6%, and 85% of patients with PVR at week 72, 96, 144, and 192, respectively. Resistance to antivirals developed in two NA-experienced patients. Failure of virological response (VR) in patients with PVR was associated with high levels of serum HBV DNA at week 48. CONCLUSIONS: Patients with PVR to ETV had favorable long-term virological outcomes. The low serum level of HBV DNA (< 200 IU/mL) at week 48 was associated with subsequent development of a VR in patients with PVR to ETV.
Adult ; Antiviral Agents/adverse effects/*therapeutic use ; Biomarkers/blood ; DNA, Viral/blood ; Drug Resistance, Viral ; Female ; Guanine/adverse effects/*analogs & derivatives/therapeutic use ; Hepatitis B virus/*drug effects/genetics/growth & development ; Hepatitis B, Chronic/diagnosis/*drug therapy ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Time Factors ; Treatment Outcome ; Viral Load

Transforming growth factor (TGF)-beta1 is a key cytokine producing extracellular matrix. We evaluated the effect of TGF-beta1 gene polymorphism at codon 10 on the development of cirrhosis in patients with chronic hepatitis B. One hundred seventy eight patients with chronic hepatitis (CH, n=57) or liver cirrhosis (LC, n=121), who had HBsAg and were over 50 yr old, were enrolled. The genotypes were determined by single strand conformation polymorphism. There were no significant differences in age and sex ratio between CH and LC groups. HBeAg positivity and detection rate of HBV DNA were higher in LC than in CH groups (P=0.055 and P=0.003, respectively). There were three types of TGF-beta1 gene polymorphism at codon 10: proline homozygous (P/P), proline/leucine heterozygous (P/L), and leucine homozygous (L/L) genotype. In CH group, the proportions of P/P, P/L, and L/L genotype were 32%, 51%, and 17%, respectively. In LC group, the proportions of those genotypes were 20%, 47%, and 33%, respectively. The L/L genotype was presented more frequently in LC than in CH groups (P=0.017). Multivariate logistic regression analysis confirms that detectable HBV DNA (odds ratio [OR]: 3.037, 95% confidence interval [CI]: 1.504-6.133, P=0.002) and L/L genotype (OR: 3.408, 95% CI: 1.279-9.085, P=0.014) are risk factors for cirrhosis.
Aged ; Asian Continental Ancestry Group/genetics ; *Carrier State ; *Codon ; Female ; Genetic Predisposition to Disease ; Genotype ; Hepatitis B virus/genetics ; *Hepatitis B, Chronic/genetics/virology ; Humans ; *Liver Cirrhosis/genetics/virology ; Male ; Middle Aged ; Odds Ratio ; *Polymorphism, Genetic ; Risk Factors ; Transforming Growth Factor beta1/*genetics

BACKGROUNDS/AIMS: Hepatitis B virus (HBV) replicates via RNA intermediates, which could serve as targets for RNA interference (RNAi). Vector-mediated short-hairpin RNA (shRNA) can induce sustained RNAi in comparison to small interfering RNA. Lentiviral vector is known to induce prolonged RNAi with high transduction efficiency. In this study, we sought to test the in vitro efficacy of shRNA delivered by a lentiviral vector in suppressing the replication of HBV. METHODS: Two shRNA sequences against the hepatitis B viral protein HBx (sh1580 and sh1685) were cloned downstream of the U6 promoter in an HIV-based plasmid to generate third-generation lentiviral vectors. HepAD38 cells were transduced with anti-HBx lentiviral vectors, and HBV replication was induced for 5 days. HBV DNA was isolated and quantified using real-time PCR. RESULTS: Lentiviral vectors encoding the shRNA against HBV transduced HepAD38 cells with high efficacy. The total intracellular HBV DNA content was significantly reduced by both sh1580 and sh1685 (2.9% and 12.0%, respectively; P<0.05). HBV covalently closed circular DNA (cccDNA) was also suppressed significantly (19.7% and 25.5%, respectively; P<0.05). CONCLUSIONS: Lentivirus-mediated delivery of shRNA against HBx can effectively suppress the replication of HBV and reduce HBV cccDNA in cell culture systems.
Cell Line, Tumor ; Genetic Vectors ; Hepatitis B virus/*genetics/growth & development/physiology ; Humans ; Lentivirus/*genetics ; *RNA Interference ; RNA, Viral/metabolism ; Trans-Activators/*antagonists & inhibitors/genetics/metabolism ; *Virus Replication

No abstract available.
Genetic Vectors ; Hepatitis B virus/*genetics/growth & development/physiology ; Humans ; Lentivirus/genetics ; *RNA Interference ; RNA, Viral/metabolism ; Trans-Activators/antagonists & inhibitors/genetics ; *Virus Replication

<b>BACKGROUNDb>MicroRNAs (miRNAs) are highly conserved small non-coding RNAs of 18 - 25 nucleotides (nt) that mediate post-transcriptional gene regulation. Hepatitis B virus (HBV) can cause either acute or chronic hepatitis B, and is a high risk factor for liver cirrhosis and hepatocellular carcinoma. Some mammalian viruses have been shown to modulate the expression of host cellular miRNAs. However, interactions between the HBV and the host cellular miRNAs are largely unknown.

<b>METHODSb>miRNA microarray and Northern blotting analysis were used to compare the expression profile of cellular miRNAs of a stable HBV-expressing cell line HepG2.2.15 and its parent cell line HepG2. mRNA microarray assay and the miRanda program were used to predict the miRNA targets. A flow cytometric assay was further used to investigate the expression of human leukocyte antigen (HLA)-A.

<b>RESULTSb>Eighteen miRNAs were differentially expressed between the two cell lines. Among them, eleven were up-regulated and seven were down-regulated in HepG2.2.15 cells. Northern blotting analysis confirmed that the expression of miR-181a, miR-181b, miR-200b and miR-146a were up-regulated and the expression of miR-15a was down-regulated, which was in consistent with the results of the microarray analysis. Furthermore, some putative miRNA targets were predicted and verified to be linked with mRNA expression. The 3'-UTR of HLA-A gene had one partially complementary site for miR-181a and miR-181a might down-regulate the expression of HLA-A.

<b>CONCLUSIONb>HBV replication modulates the expression of host cellular miRNAs, which may play a role in the pathogenesis of HBV-related liver diseases.

Blotting, Northern ; Cell Line, Tumor ; metabolism ; virology ; Flow Cytometry ; Gene Expression Profiling ; Gene Expression Regulation ; HLA-A Antigens ; metabolism ; Hepatitis B virus ; growth & development ; physiology ; Humans ; MicroRNAs ; genetics ; Oligonucleotide Array Sequence Analysis

Animals ; Carcinoma, Hepatocellular ; genetics ; metabolism ; virology ; Genes, p53 ; Hepatitis B ; genetics ; metabolism ; virology ; Hepatitis B virus ; Humans ; Inhibitor of Growth Protein 1 ; Intracellular Signaling Peptides and Proteins ; metabolism ; Liver Neoplasms ; genetics ; metabolism ; virology ; Loss of Heterozygosity ; Nuclear Proteins ; metabolism ; Point Mutation ; Signal Transduction ; Trans-Activators ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Tumor Suppressor Proteins ; metabolism

<b>OBJECTIVEb>To investigate the characteristics of circulating type II pre-dendritic cells (pDC2) and evaluate its role in patients with chronic hepatitis B virus infection.

<b>METHODSb>The quantitative alterations of pDC2 in 27 chronic HBV-infected patients as treated group and 15 healthy individuals as a control group were analyzed by using flow cytometry based on the comparison of CD4+/CD8+ ratios of T lymphocyte subsets between the two groups. The IFN-alpha-producing ability of pDC2 after incubation was determined by ELISA.

<b>RESULTSb>The percentage of pDC2 (0.096 +/- 0.086) from the peripheral blood in chronic HBV-infected patients were significantly lower than that (0.304 +/- 0.093) from the normal controls (P less than 0.001) while the CD4+/CD8+ ratios were higher than those in normal controls (P less than 0.01). The values of IFN-alpha-producing function and IL-12 of circulating pDC2 in chronic HBV-infected patients group were significantly lower than those in healthy subjects (P < 0.001). The percentage of pDC2 and CD4+/CD8+ ratios were higher in the patients positive for HBV DNA in sera than those in patients negative for HBV DNA in sera (P < 0.01).

<b>CONCLUSIONb>The decreased number of circulating pDC2 and IFN-alpha-producing function from peripheral blood in patients with chronic hepatitis B virus infection may result in the decline of host immune response, which may partially contribute to the disease progress of HBV infection and existence of viral genomic DNA in patient's sera.

Adolescent ; Adult ; CD4-CD8 Ratio ; Cell Count ; DNA, Viral ; blood ; genetics ; Dendritic Cells ; cytology ; immunology ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Hepatitis B virus ; genetics ; growth & development ; Hepatitis B, Chronic ; blood ; immunology ; virology ; Humans ; Interleukin-12 ; biosynthesis ; Male ; Middle Aged ; T-Lymphocyte Subsets ; cytology ; immunology ; Young Adult

<b>OBJECTIVEb>To establish a culture system of HBV positive serum infected Hep G2 cells in vitro.

<b>METHODSb>Hep G2 cells were seeded into six-well cluster dishes, at 1 x 10(-6) cells per well and incubated with 3 ml 10% fetal calf serum/ Dulbecco's modified Eagle's medium (10% FCS/DMEM) at 37 degrees in 5% CO2 air. At 24 h after plating, infection group Hep G2 cells were cultured with 0.5 ml HBV positive serum, in control group HBV negative serum was used, 24 h later the inoculums was removed. The cells were then extensively washed with 0.01 mol/L phosphate-buffered saline (PBS). After washing with PBS, 4 ml 2% FCS/DMEM were added to each well and the medium was collected every 12 h. ELISA method was used to detect HBsAg in culture medium. HBV DNA in cells and culture medium was detected by PCR.

<b>RESULTSb>In infection group, HBsAg could be detected from cell culture medium from 12 h (after PBS washed) to 84 h. HBV DNA could be detected by PCR in culture medium and cells.

<b>CONCLUSIONb>Infection of Hep G2 cells by HBV positive serum is feasible.

Carcinoma, Hepatocellular ; pathology ; virology ; Cell Line, Tumor ; DNA, Viral ; genetics ; Enzyme-Linked Immunosorbent Assay ; Hepatitis B ; blood ; virology ; Hepatitis B Surface Antigens ; analysis ; Hepatitis B virus ; genetics ; growth & development ; immunology ; Humans ; Liver Neoplasms ; pathology ; virology ; Polymerase Chain Reaction ; Serum ; virology