Stable HBV-producing Cell Line Research Articles

HBx-induced S100A9 in NF-\u03baB dependent manner promotes growth and metastasis of hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is associated with hepatitis B virus (HBV) infection. Myeloid-specific S100 proteins (S100s), namely, S100A8, S100A9 and S100A12, have been recently recognized as newly discovered damage-associated molecular patterns (DAMPs) that are correlated with progression in pathogen of infectious diseases. However, whether S100s are regulated by HBV and involved in HBV-related hepatocarcinogenesis are still unclear. Here, we found that all expression levels of myeloid-specific S100s (S100A8, S100A9 and S10012) were elevated in serum and tissue samples from HCC patients. Expression of S100A9 but not S100A8 and S10012 were also higher in blood serum and tissue samples from HBV-positive HCC patients than that in HBV-negative HCC patients. High levels of intracellular and extracellular S100A9 were also confirmed in HepG2 cells expressing 1.3-fold HBV genome or HBV-encoded X protein (HBx) as well as in a stable HBV-producing cell line HepG2.2.15. HBx was shown to facilitate translocation of NF-κB from the cytoplasm to the nucleus, and NF-κB bound to the promoter of S100A9 to enhance its transcription. Silencing S100A9 expression partially blocked HBx-induced growth and metastasis of HepG2 cells both in vitro and in vivo. Further, serum S100A9 levels were found to correlate with TNM stage, extrahepatic metastasis status and HBV DNA load in HBV-related HCC and also had a better diagnostic value for identifying extrahepatic metastasis. Our these data demonstrate that S100A9 plays a pivotal role in HBx-induced growth and metastasis of HCC and may serve as a potential diagnostic marker for extrahepatic metastasis.

Novel stable HBV producing cell line systems for expression and screening antiviral inhibitor of hepatitis B virus in human hepatoma cell line

Chronic hepatitis B virus (HBV) infection is currently a major public health burden. Therefore, there is an urgent need for the development of novel antiviral inhibitors. The stable HBV-producing cell lines of genotype D are widely used to investigate the HBV life cycle and to evaluate antiviral agents. However, stable HBV-producing cell lines of different genotypes do not exist. To construct more convenient and efficient novel cell systems, stable cell lines of genotypes A, B, and C were established using a full-length HBV genome sequence isolated from chronic HBV patients in human hepatoma HepG2 cells. Novel HBV clones were identified and stable HBV-producing cell lines derived from these clones were constructed. HBV replication activities demonstrated time-dependent expression, and the novel cell lines were susceptible to several antiviral inhibitors with no cytotoxicity. Furthermore, infectious viruses were produced from these cell lines. In conclusion, we have established novel stable HBV-producing cell line systems of genotypes A, B, and C. These systems can provide valuable tools for screening antiviral agents and analyzing viral phenotypes in vitro.

MiR-200c targets nuclear factor IA to suppress HBV replication and gene expression via repressing HBV Enhancer I activity

BackgroundHepatitis B virus (HBV) chronic infection is a health problem in the worldwide, with a underlying higher risk of liver cirrhosis and hepaticocellular carcinoma. A number of studies indicate that microRNAs (miRNAs) play vital roles in HBV replication. This study was designed to explore the potential molecular mechanism of miR-200c in HBV replication. MethodsThe expression of miR-200c, nuclear factor IA (NFIA) mRNA, HBV DNA, and HBV RNA (pregenomic RNA (pgRNA), and total RNA) were measured by qRCR. The levels of HBsAg and HBeAg were detected by ELISA. NFIA expression at protein level was measured by western blot. The direct interaction between miR-200c and NFIA were identified by Targetscan software and Dual-Luciferase reporter analysis. Enhance I activity were detected by Dual-Luciferase reporter assay. ResultsmiR-200c expression was prominently reduced in pHBV1.3-tranfected Huh7 and in stable HBV-producing cell line (HepG2.2.15). The enforced expression of miR-200c significantly suppressed HBV replication, as demonstrated by the reduced levels of HBV protein (HBsAg and HBeAg) and, DNA and RNA (pgRNA and total RNA) levels. NFIA was proved to be a target of miR-200c and NFIA overexpression notably stimulated HBV replication. In addition, the inhibitory effect of miR-200c on HBV Enhance I activity was abolished following restoration of NFIA. ConclusionsmiR-200c repressed HBV replication by directly targeting NFIA, which might provide a novel therapeutic target for HBV infection.

An Effective Molecular Target Site in Hepatitis B Virus S Gene for Cas9 Cleavage and Mutational Inactivation.

Chronic hepatitis B infection remains incurable because HBV cccDNA can persist indefinitely in patients recovering from acute HBV infection. Given the incidence of HBV infection and the shortcomings of current therapeutic options, a novel antiviral strategy is urgently needed. To inactivate HBV replication and destroy the HBV genome, we employed genome editing tool CRISPR/Cas9. Specifically, we found a CRISPR/Cas9 system (gRNA-S4) that effectively targeted the HBsAg region and could suppress efficiently viral replication with minimal off-target effects and impact on cell viability. The mutation mediated by CRISPR/Cas9 in HBV DNA both in a stable HBV-producing cell line and in HBV transgenic mice had been confirmed and evaluated using deep sequencing. In addition, we demonstrated the reduction of HBV replication was caused by the mutation of S4 site through three S4 region-mutated monoclonal cells. Besides, the gRNA-S4 system could also reduce serum surface-antigen levels by 99.91 ± 0.05% and lowered serum HBV DNA level below the negative threshold in the HBV hydrodynamics mouse model. Together, these findings indicate that the S4 region may be an ideal target for the development of innovative therapies against HBV infection using CRISPR/Cas9.

Antihepatitis B Virus Activity of a Protein-Enriched Fraction from Housefly (Musca domestica) in a Stable HBV-Producing Cell Line

Hepatitis B virus (HBV) infection remains a major public health problem. Although several vaccines and therapeutic strategies are currently being implemented to combat HBV virus, effective antiviral therapy against HBV infection has not been fully developed. Alternative strategies and new drugs to combat this disease are urged. Insects and insect derivatives are a large and unexploited source of potentially useful compounds for modern medicine. In the present study, we investigated the first anti-HBV activity of a protein-enriched fraction (PE) from the larvae of the housefly (Musca domestica) in a stable HBV-producing cell line. HBsAg and HBeAg in the culture medium were measured by enzyme-linked immunosorbent assay. HBV-DNA was quantified by fluorescent quantification PCR. HBV core protein was assayed by immunofluorescent staining. Results indicate PE treatment inhibited both HBsAg, HBeAg secretion, and HBV-DNA replication. Furthermore, PE could also suppress HBV core protein expression. PE could be a potential candidate for the development of a novel and effective drug for the treatment of HBV infection.

MicroRNA-501 promotes HBV replication by targeting HBXIP

MicroRNAs (miRNAs) can negatively regulate gene expression and also induce or inhibit viral replication. In the present study, we found 10 miRNAs were differentially expressed in a stable HBV-producing cell line (HepG2.2.15) compared with its control cell line (HepG2) by miRNA array analysis. miR-501 was significantly up-regulated in HepG2 cells and tissues with high-HBV replication. miR-501 expression was significantly up-regulated in hepatocellular carcinoma tissues, where HBV replication kept high. Down-regulating miR-501 could significantly inhibit HBV replication, but not influence the growth of HepG2.2.15 cells. Luciferase reporter and western blot assays revealed that HBXIP, an inhibitor of HBV replication, was a potential target of miR-501. Moreover, knockdown of HBXIP rescued the inhibition of HBV that occurred after the loss of miR-501 in HepG2.2.15 cells, suggesting that miR-501 induced HBV replication partially by targeting HBXIP. Thus, knockdown of miR-501 might provide a new mechanism and therapeutic target for inhibiting HBV replication.

48 DEMOGRAPHIC, CLINICAL, BIOCHEMICAL, RADIOLOGICAL, AND ETIOLOGICAL PROFILE AND THE RISK FACTORS ASSOCIATED WITH 357 HEPATOCELLULAR CARCINOMA CASES FROM INDIA

S 20TH ANNUAL CONFERENCE OF INASL, MARCH 2–4, 2012 S28 © 2012, INASL A b tra ts in various signal transduction pathways leading to progression in hepatocarcinogenesis. Aim: To investigate the possible role of HBx in the modulation of microRNA-222 expression associated in HBVrelated HCC. Method: HepG2 cells were transiently transfected with pCXN2-HBx plasmid (D genotype), and stable HBVproducing cell line HepG2.2.15 was used as model for chronic HBV infection in vitro system. Expression of microRNA-222, its target mRNA p27 was analyzed by real-time polymerase chain reaction (PCR). Western blot was used for target protein p27 expression. Result: Our results demonstrated that miR-222 was downregulated in both stably HBV replicating HepG2.2.15 cell line and transiently transfected HepG2 cells by X plasmid compared to HepG2 cells transfected by empty expression vector (pCXN2). The expression of target mRNA and target protein p27 was found to be up-regulated accordingly in both HBV stable cell line and upon transient transfection of HepG2 cells by X plasmid. Further, expression of miRNAs and their target mRNAs was modulated with dose dependant manner during transient transfection of X gene in HepG2 cells. Conclusion: Hepatitis B virus X protein induced modulation in the expression pattern of miRNA-222.

49 HEPATITIS B VIRUS X PROTEIN INDUCED MODULATION IN THE EXPRESSION OF MIR-222 IN MALIGNANT HEPATOCYTES IN VITRO

S 20TH ANNUAL CONFERENCE OF INASL, MARCH 2–4, 2012 S28 © 2012, INASL A b tra ts in various signal transduction pathways leading to progression in hepatocarcinogenesis. Aim: To investigate the possible role of HBx in the modulation of microRNA-222 expression associated in HBVrelated HCC. Method: HepG2 cells were transiently transfected with pCXN2-HBx plasmid (D genotype), and stable HBVproducing cell line HepG2.2.15 was used as model for chronic HBV infection in vitro system. Expression of microRNA-222, its target mRNA p27 was analyzed by real-time polymerase chain reaction (PCR). Western blot was used for target protein p27 expression. Result: Our results demonstrated that miR-222 was downregulated in both stably HBV replicating HepG2.2.15 cell line and transiently transfected HepG2 cells by X plasmid compared to HepG2 cells transfected by empty expression vector (pCXN2). The expression of target mRNA and target protein p27 was found to be up-regulated accordingly in both HBV stable cell line and upon transient transfection of HepG2 cells by X plasmid. Further, expression of miRNAs and their target mRNAs was modulated with dose dependant manner during transient transfection of X gene in HepG2 cells. Conclusion: Hepatitis B virus X protein induced modulation in the expression pattern of miRNA-222.

50 ACETIC ACID ABLATIVE THERAPY FOR THE TREATMENT OF HEPATOCELLULAR CARCINOMA-A PROMISING AND AFFORDABLE OPTION

S 20TH ANNUAL CONFERENCE OF INASL, MARCH 2–4, 2012 S28 © 2012, INASL A b tra ts in various signal transduction pathways leading to progression in hepatocarcinogenesis. Aim: To investigate the possible role of HBx in the modulation of microRNA-222 expression associated in HBVrelated HCC. Method: HepG2 cells were transiently transfected with pCXN2-HBx plasmid (D genotype), and stable HBVproducing cell line HepG2.2.15 was used as model for chronic HBV infection in vitro system. Expression of microRNA-222, its target mRNA p27 was analyzed by real-time polymerase chain reaction (PCR). Western blot was used for target protein p27 expression. Result: Our results demonstrated that miR-222 was downregulated in both stably HBV replicating HepG2.2.15 cell line and transiently transfected HepG2 cells by X plasmid compared to HepG2 cells transfected by empty expression vector (pCXN2). The expression of target mRNA and target protein p27 was found to be up-regulated accordingly in both HBV stable cell line and upon transient transfection of HepG2 cells by X plasmid. Further, expression of miRNAs and their target mRNAs was modulated with dose dependant manner during transient transfection of X gene in HepG2 cells. Conclusion: Hepatitis B virus X protein induced modulation in the expression pattern of miRNA-222.

In vitro anti-hepatitis B virus effect of Hypericum perforatum L.

The anti-hepatitis B virus (HBV) effects and its mechanisms of the ethanol extracts of Hypericum perforatum L. (EHP) in vitro were explored. HepG2 2.2.15 cells, a stable HBV-producing cell line, were cultured as the model system to observe the anti-HBV effect. The viral antigens of cellular secretion, HBsAg and HBeAg, were determined by enzyme linked immunosorbent assay (ELISA). The quantity of HBV-DNA released in the supernatant was assayed by real-time PCR. In order to understand the mechanisms of the suppression of HBV replication, all HBV promoters (Cp, Xp, S1p, S2p and Fp) with luciferase reporter gene were transfected into HepG2 cells respectively. Then the activities of viral promoters were examined by luciferase reporter assay. It was found EHP effectively suppressed the secretion of HBsAg and HBeAg from HepG2 2.2.15 cells in a dose-dependent manner, as well as the extracellular HBV DNA. And EHP could selectively inhibit the activity of HBV promoter Fp. Our data suggest that EHP exerts anti-HBV effects via inhibition of HBV transcription, which helps to elucidate the mechanism underlying the potential therapeutic value of EHP.

Identification of Natural Compounds with Anti-Hepatitis B Virus Activity from Rheum palmatum L. Ethanol Extract

Background: Hepatitis B virus (HBV) infection is a severe health problem in the world; however, there is still no satisfactory therapeutic strategy for the HBV infection. In search for new anti-HBV agents with higher efficiency and less side effects, the anti-HBV activities of traditional Chinese medicine Rheum palmatum L. ethanol extract (RPE) and isolated anthraquinones were evaluated. Methods: The anti-HBV activities of RPE and isolated anthraquinones were demonstrated in a stable HBV-producing cell line HepG2 2.2.15 by using real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and Southern blot analysis. Results: RPE could inhibit HBV-DNA production and HBsAg expression in a dose-dependent manner. The concentration of 50% HBV-DNA inhibition (IC₅₀) of RPE was calculated at 212.36 ± 11 µg/ml. Six anthraquinones were isolated from RPE by using RP-HPLC. Five free anthraquinones showed weakly or slightly inhibitory activities against HBV. The only combined anthraquinone chrysophanol 8-O-β-D-glucoside exhibited significant activity against HBV DNA production and antigens expression with an IC₅₀ value of 36.98 ± 2.28 µg/ml on HBV DNA inhibition. Endogenous HBV DNA polymerase activity assay indicated that chrysophanol 8-O-β-D-glucoside might be a potential inhibitor of the HBV DNA polymerase. Conclusions: The results suggested that RPE could effectively inhibit HBV. The combined anthraquinone chrysophanol 8-O-β-D-glucoside is the major active compound in RPE and could be a promising candidate for the development of new anti-HBV drugs in the treatment of HBV infection.

Inhibition of hepatitis B virus replication by Rheum palmatum L. ethanol extract in a stable HBV-producing cell line

Hepatitis B virus(HBV) infection is a severe health problem in the world. However, there is still not a satisfactory therapeutic strategy for the HBV infection. To search for new anti-HBV agents with higher efficacy and less side-effects, the inhibitory activities of traditional Chinese medicine Rheum palmatum L. ethanol extract(RPE) against HBV replication were investigated in this study. Quantitative real-time polymerase chain reaction(PCR) was employed to analyze the inhibitory activity of RPE against HBV-DNA replication in a stable HBV-producing cell line HepAD38; the expression levels of HBV surface antigen(HBsAg) and e antigen(HBeAg) were also determined by enzyme linked immunosorbent assay(ELISA) after RPE treatment. RPE could dose-dependently inhibit the production of HBV-DNA and HBsAg. The concentration of 50% inhibition(IC50) was calculated at 209.63, 252.53 μg /mL, respectivel y. However, its inhibitory activity against HBeAg expression was slight even at high concentrations. RPE had a weak cytotoxic effect on HepAD38 cells(CC50 = 1 640 μg /mL) and the selectivity index(SI) was calculated at 7.82. Compared with two anthraquinone derivatives emodin and rhein, RPE showed higher ability of anti-HBV and weaker cytotoxicity. So Rheum palmatum L. might possess other functional agents which could effectively inhibit HBV-DNA replication and HBsAg expression. Further purification of the active agents, identification and modification of their structures to improve the efficacy and decrease the cytotoxicity are required.

Inhibition of human La protein by RNA interference downregulates hepatitis B virus mRNA in 2.2.15 cells.

To investigate the role of human La protein in HBV mRNA expression. Three human La protein (hLa) specific siRNA expression cassettes (SECs) containing U6+1 promoter were prepared via one-step overlapping extension PCR. After transfection with SECs into HepG2 cells, inhibition effects on hLa expression were analyzed by semi-quantitative RT-PCR and Western blotting. Then, effective SECs were screened out and transfected into 2.2.15 cells, a stable HBV-producing cell line. HBV surface antigen (HBsAg) and e antigen (HBeAg) secretions into culture media were detected by microparticle enzyme immunoassay (MEIA) and HBs and HBe mRNA levels were analyzed by semi-quantitative RT-PCR. SEC products containing U6+1 snRNA promoter, and 3 sites of hLa mRNA specific siRNA were obtained successfully by one-step overlapping extension PCR and could be directly transfected into HepG2 cells, resulting in inhibition of La protein expression in both mRNA and protein levels, among which U6+1-hLa833 was the most efficient, which reduced 18.6-fold mRNA and 89% protein level respectively. In 2.2.15 cells, U6+1-hLa833 was also efficient on inhibition of hLa expression. Furthermore, semi-quantitative RT-PCR showed that HBs and HBe mRNA levels were significantly decreased by 8- and 66-fold in U6+1-hLa833 transfected cells compared to control. Accordingly, HBsAg and HBeAg secretions were decreased partly posttransfection with SECs. PCR-based SECs can be used to mediate RNAi in mammalian cells and provide a novel approach to study the function of La protein. The inhibition of La protein expression can result in a significant decrease of HBV mRNA, which implies that the hLa protein is also involved HBV RNA metabolism as one of the HBV RNA-stabilizing factors in human cells.

Inhibition of HBV replication by siRNA in a stable HBV-producing cell line

Potent inhibition of endogenous gene expression by RNA interference has been achieved by using sequence-specific posttranscriptional gene silencing through the action of small interfering RNA molecules (siRNA). In these reports, the natural function of genes could be deduced through the ensuing loss of function. Based on the extraordinary effectiveness in silencing endogenous genes, we wondered whether siRNA could be applied against viral replication in a hepatitis B virus (HBV) model using HBV-specific siRNA. To test this idea, HepG2 2.2.15, a human hepatoblastoma cell line that constitutively produces infectious HBV particles, was transfected with HBV-specific siRNAs and controls. HBV surface antigen (HBsAg) secretion into culture media was inhibited by 78%, 67%, and 42% with siRNA against the polyadenylation (PA), precore (PreC), and surface (S) regions, respectively, compared with controls as detected by enzyme-linked immunosorbent assay. After exposure to HBVPA siRNA, Northern blot analysis showed that HBV pregenomic RNA levels were decreased by 72%, and levels of HBV RNA containing the polyadenylation signal sequence were suppressed by 86%, as detected by RNase protection assay. Levels of HBV core-associated DNA, a replication intermediate, also decreased by 71%. Immunocytochemistry revealed that 30% to 40% of the cells transfected with HBVPA siRNA were completely negative for detectable HBsAg levels. Controls consisting of treatment with HBV-specific siRNA alone, lipofection reagent alone, or random double-stranded RNA (dsRNA) lipofection complex failed to decrease HBV surface antigen, HBV messenger RNA (mRNA), or core-associated HBV-DNA levels. In conclusion, siRNA inhibits hepatitis B viral replication in a cell culture system. Future studies are needed to explore the specific delivery of siRNA to liver cells in vivo and the applicability of this approach. (H epatology 2003;38:842-850.)