RESUMO
More than 250 million people worldwide have chronic hepatitis B virus (HBV) infections, resulting in over 1 million annual fatalities because HBV cannot be adequately treated with current antivirals. Hepatocellular carcinoma (HCC) risk is elevated in the presence of the HBV. Novel and powerful medications that specifically target the persistent viral components are needed to remove infection. This study aimed to use HepG2.2.15 cells and the rAAV-HBV1.3 C57BL/6 mouse model established in our laboratory to examine the effects of 16F16 on HBV. The transcriptome analysis of the samples was performed to examine the impact of 16F16 therapy on host factors. We found that the HBsAg and HBeAg levels significantly decreased in a dose-dependent manner following the 16F16 treatment. 16F16 also showed significant anti-hepatitis B effects in vivo. The transcriptome analysis showed that 16F16 regulated the expression of several proteins in HBV-producing HepG2.2.15 cells. As one of the differentially expressed genes, the role of S100A3 in the anti-hepatitis B process of 16F16 was further investigated. The expression of the S100A3 protein significantly decreased following the 16F16 therapy. And upregulation of S100A3 caused an upregulation of HBV DNA, HBsAg, and HBeAg in HepG2.2.15 cells. Similarly, knockdown of S100A3 significantly reduced the levels of HBsAg, HBeAg, and HBV DNA. Our findings proved that S100A3 might be a new target for combating HBV pathogenesis. 16F16 can target several proteins involved in HBV pathogenesis, and may be a promising drug precursor molecule for the treatment of HBV.
Assuntos
Carcinoma Hepatocelular , Hepatite B Crônica , Neoplasias Hepáticas , Animais , Camundongos , DNA Viral/genética , Perfilação da Expressão Gênica , Antígenos E da Hepatite B , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B , Hepatite B Crônica/tratamento farmacológico , Camundongos Endogâmicos C57BL , Transcriptoma , Humanos , Células Hep G2/metabolismo , Células Hep G2/virologia , Antivirais/farmacologiaRESUMO
Tetraspanins are master organizers of the cell membrane. Recent evidence suggests that tetraspanins themselves may become crowded by virus particles and that these crowds/aggregates co-internalize with the viral particles. Using microscopy, we studied human papillomavirus (HPV) type 16-dependent aggregates on the cell surface of tetraspanin overexpressing keratinocytes. We find that aggregates are (1) rich in at least two different tetraspanins, (2) three-dimensional architectures extending up to several micrometers into the cell, and (3) decorated intracellularly by filamentous actin. Moreover, in cells not overexpressing tetraspanins, we note that obscurin-like protein 1 (OBSL1), which is thought to be a cytoskeletal adaptor, associates with filamentous actin. We speculate that HPV contact with the cell membrane could trigger the formation of a large tetraspanin web. This web may couple the virus contact site to the intracellular endocytic actin machinery, possibly involving the cytoskeletal adaptor protein OBSL1. Functionally, such a tetraspanin web could serve as a virus entry platform, which is co-internalized with the virus particle.
Assuntos
Actinas/fisiologia , Proteínas do Citoesqueleto/fisiologia , Papillomavirus Humano 16/fisiologia , Tetraspanina 24/fisiologia , Tetraspanina 30/fisiologia , Endocitose , Células HaCaT/virologia , Células HeLa/ultraestrutura , Células HeLa/virologia , Células Hep G2/virologia , Humanos , Microscopia Confocal , Microscopia Eletrônica , Infecções por Papillomavirus/virologia , Plaquinas/fisiologia , Vírion/fisiologia , Vírion/ultraestrutura , Internalização do VírusRESUMO
The extraction conditions and chromatographic analysis from seeds of Inga edulis were optimized and provided one anthocyanin from aqueous fraction and a mixture of three anthocyanins from methanolic fraction. The pure anthocyanin obtained was subjected to structural modifications and the products obtained were subjected to chemical and pharmacological assays, as well as quantum chemical calculations based on DFT and TD-DFT methods. Hence, the anthocyanin fractions were evaluated for their chemical-pharmacological potential through chemical and biological assays: antioxidant activity by the DPPH, determination of the Solar Protection Factor (SPF) and cytotoxic activity (hepatocellular carcinoma infected with hepatitis C virus). The results indicated that even the anthocyanin and derivatized compounds having high antioxidant potential showed an SPF lower than six, which is lower than the minimum accepted by current Brazilian legislation. In addition, none of compounds presented significant cytotoxic activity against the tumour cell line studied.
Assuntos
Antocianinas/química , Antocianinas/farmacologia , Fabaceae/química , Antocianinas/análise , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Brasil , Teoria da Densidade Funcional , Frutas/química , Células Hep G2/virologia , Hepacivirus/patogenicidade , Humanos , Espectroscopia de Ressonância Magnética , Extratos Vegetais/químicaRESUMO
Dengue is a mosquito-borne disease caused by dengue virus (DENV) infection. There is currently no effective vaccine or antiviral treatment available against DENV. In previous studies, we showed that Lactococcus lactis strain Plasma (LC-Plasma) could activate plasmacytoid dendritic cells, which play an important role against virus infection. LC-Plasma administration ameliorated symptoms of viral diseases and its effect appeared to be associated with IFN-α induction. However the precise mechanism of LC-Plasma protection remained unclear. In this study, we investigated the effects of LC-Plasma-induced humoral factors on DENV replication using HepG2 cells as an in vitro infection model. When HepG2 cells were preincubated with supernatants of LC-Plasma-stimulated bone marrow-derived dendritic cells, the replication of DENV was significantly inhibited in a dose dependent manner and its activity was evident regardless of the DENV serotype. In addition, the expression of interferon-stimulated genes, including ISG15, IFITM-1, MxA, RSAD2, and RyDEN, was significantly upregulated by humoral factors. We also compared the effects of representative strains of lactic acid bacteria and found that the ability to prevent DENV replication was unique to LC-Plasma. In addition, it was revealed that both anti-DENV replication activity and ISG induction depended on type I IFN rather than type III IFN signaling. Taken together, since LC-Plasma induces, in a more natural form, potent anti-DENV replication activities irrespective of viral serotypes via induction of type I IFN, LC-Plasma could be safely used as a prophylactic anti-DENV option.
Assuntos
Células Dendríticas/imunologia , Vírus da Dengue/crescimento & desenvolvimento , Células Hep G2/virologia , Imunidade Inata , Fatores Imunológicos/metabolismo , Lactococcus lactis/imunologia , Plasma/imunologia , Animais , Linhagem Celular , Vírus da Dengue/imunologia , Humanos , Camundongos Endogâmicos BALB C , Replicação Viral/imunologiaRESUMO
Hepatitis B virus (HBV) infection is a major global health burden as chronic hepatitis B (CHB) is associated with the development of liver diseases including hepatocellular carcinoma (HCC). To gain insight into the mechanisms causing HBV-related HCC, we investigated the effects of HBV replication on global host cell gene expression using human HepG2 liver cells. By microarray analysis, we identified 54 differentially expressed genes in HBV-replicating HepG2 cells. One of the differentially-expressed genes was insulin-like growth factor binding protein 1 (IGFBP1) which was downregulated in HBV-replicating cells. Consistent with the gene expression data, IGFBP1 was suppressed at both the cellular and secreted protein levels in the presence of HBV replication. Transient transfection experiments with an inducible plasmid encoding the HBV X protein (HBx) revealed that HBx alone was sufficient to modulate IGFBP1 expression. Small interference RNA (siRNA)-mediated loss of function studies revealed that knockdown of IGFBP1 reduced apoptosis induced by either thapsigargin (TG) or staurosporine (STS). Treatment of cells with recombinant insulin-like growth factor 1 (IGF-1) decreased both TG- or STS-induced apoptosis. Interestingly, addition of recombinant IGFBP1 reversed the anti-apoptotic effect of IGF-1 on TG-induced, but not STS-induced, apoptosis. In conclusion, our results suggest an anti-apoptotic autocrine function of HBV-mediated downregulation of IGFBP1 in HepG2 cells. Such an effect may contribute to the development of HBV-mediated HCC by increasing pro-survival and anti-apoptotic IGF-1 effects.
Assuntos
Apoptose/fisiologia , Carcinoma Hepatocelular/virologia , Células Hep G2/virologia , Vírus da Hepatite B/patogenicidade , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Regulação para Baixo , Hepatite B/virologia , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Transativadores/metabolismo , Proteínas Virais Reguladoras e AcessóriasRESUMO
Hepatitis C is a serious health issue and cause liver disorders in millions of people. Available therapeutic agents require long term administration with numerous side effects. Therefore, there is a dire need to find alternative treatment options for this disease. Since ancient times, medicinal plants are widely used to cure various diseases with no or less harmful effects. Therefore, this study was designed to find out phytochemicals and investigate antiviral activity of methanol extract of Ajuga bracteosa, Ajuga parviflora, Berberis lycium and Citrus lemon against Hepatitis C Virus (HCV infection). Phytochemical analysis of the plant extract was performed using various chemical tests. Toxicity of the plant extract was determined against using trypan blue exclusion method. Antiviral activity of the selected plant extract was find out against HCV infected HepG2 cells. For this purpose, HepG2 cells were seeded with HCV positive and negative serum and nontoxic doses of plant extract for 24 and 48â¯h. After this RNA was extracted and viral load was determined using Real-time PCR. Phytochemical analysis showed the presence of flavonoids and phenols in all plant extracts while amino acids, alkaloids and tannins were present in B. lycium and saponins were detected in C. lemon. Toxicity assay showed that all plant extracts were nontoxic at maximum concentration of 200⯵g/ml except B. lycium, which showed mild toxicity at 40⯵g/ml and were extremely toxic at 60⯵g/ml and above doses. Real-time PCR quantitation result revealed that after 24â¯h treatments A. parviflora showed highest antiviral activity, followed by A. bracteosa, while B. lycium extract had low (35%) and C. lemon has no antiviral effects. The 48â¯h treatments showed an increase antiviral activity by A. bracteosa followed by A. parviflora and B. lycium while C. lemon showed negative effect. Our results depicted that mentioned plants might be used as an alternative therapeutic regime or in combination with existing treatments against HCV.
Assuntos
Ajuga/química , Antivirais/farmacologia , Berberis/química , Citrus/química , Hepacivirus/efeitos dos fármacos , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/farmacologia , Adulto , Idoso , Alcaloides/análise , Aminoácidos/análise , Proliferação de Células/efeitos dos fármacos , Feminino , Flavonoides/análise , Células Hep G2/efeitos dos fármacos , Células Hep G2/virologia , Hepatite C/tratamento farmacológico , Hepatócitos/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Fenóis/análise , Extratos Vegetais/química , Plantas Medicinais/química , Taninos/análise , Carga Viral , Adulto JovemRESUMO
Infection with the mosquito transmitted dengue virus (DENV) remains a significant worldwide public health problem. While the majority of infections are asymptomatic, infection can result in a range of symptoms. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through repression or degradation of mRNAs. To understand the contribution of miRNAs to DENV 2 replication, we screened a number of candidate miRNAs for variations in expression levels during DENV 2 infection of HepG2 (liver) cells. Seven miRNAs were identified as differentially expressed, and one, miR-21, was differentially expressed at all time points examined. Interestingly, miR-21 was also differentially regulated in DENV 2 infection under conditions of antibody dependent enhancement of infection, and in direct Zika virus infection, but not in DENV 4 infection. The role of miR-21 during DENV infection was further examined by treating HepG2 cells with an anti-miR-21 (AMO-21) before DENV infection. The results showed a significant reduction in DENV 2 production, clearly suggesting that miR-21 plays a key role in DENV 2 replication. To further confirm the role of miR-21 in DENV infection, a peptide nucleic acid-21 (PNA-21) construct with a nucleotide sequence complementary to AMO-21, was co-administered with AMO-21 as an AMO-21/PNA-21 complex followed by DENV 2 infection. The results showed that AMO-21 significantly reduced DENV 2 titer, PNA-21 significantly increased DENV 2 titer and the combined AMO-21/PNA-21 showed no difference from non-treated infection controls. Taken together, the results show that miR-21 promotes DENV 2 replication, and this mechanism could serve as a possible therapeutic intervention point.
Assuntos
Vírus da Dengue/efeitos dos fármacos , Células Hep G2/efeitos dos fármacos , Células Hep G2/virologia , MicroRNAs/farmacologia , Replicação Viral/efeitos dos fármacos , Células A549 , Antagomirs/farmacologia , Proteínas de Transporte , Replicação do DNA/efeitos dos fármacos , Dengue/virologia , Vírus da Dengue/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Humanos , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Ácidos Nucleicos Peptídicos/farmacologia , Sorogrupo , Células U937RESUMO
Dengue is an important mosquito borne viral disease in the world. Dengue virus (DENV) encodes a polyprotein, which is cleaved in ten proteins, including the non-structural protein 1 (NS1). In this work, we analyzed the effect of NS1 expression in one hepatic cell line, HepG2, through a shotgun proteomic approach. Cells were transfected with pcENS1 plasmid, which encodes the DENV2 NS1 protein, or the controls pcDNA3 (negative control) and pMAXGFP (GFP, a protein unrelated to dengue). Expression of NS1 was detected by immunofluorescence, western blot and flow cytometry. We identified 14,138 peptides that mapped to 4,756 proteins in all analyzed conditions. We found 41 and 81 differentially abundant proteins when compared to cells transfected with plasmids pcDNA3 and pMAXGFP, respectively. Besides, 107 proteins were detected only in the presence of NS1. We identified clusters of proteins involved mainly in mRNA process and viral RNA replication. Down regulation expression of one protein (MARCKS), identified by the proteomic analysis, was also confirmed by real time PCR in HepG2 cells infected with DENV2. Identification of proteins modulated by the presence of NS1 may improve our understanding of its role in virus infection and pathogenesis, contributing to development of new therapies and vaccines. BIOLOGICAL SIGNIFICANCE: Dengue is an important viral disease, with epidemics in tropical and subtropical regions of the world. The disease is complex, with different manifestations, in which the liver is normally affected. The NS1 is found in infected cells associated with plasma membrane and secreted into the circulation as a soluble multimer. This protein is essential for virus viability, although its function is not elucidated. Some reports indicate that the NS1 can be used as a protective antigen for the development of a dengue vaccine, while others suggest its involvement in viral pathogenesis. In this work, we report an in-depth comprehensive proteomic profiling resulting from the presence of NS1 in HepG2 cells. These results can contribute to a better understanding of the NS1 role during infection.
Assuntos
Proteômica/métodos , Proteínas não Estruturais Virais/fisiologia , Análise por Conglomerados , Vírus da Dengue/química , Vírus da Dengue/fisiologia , Células Hep G2/virologia , Interações Hospedeiro-Patógeno , Humanos , Fígado/virologia , RNA Mensageiro/análise , RNA Viral/análise , Transfecção , Proteínas não Estruturais Virais/análise , Proteínas não Estruturais Virais/genética , Proteínas Virais/análise , Proteínas Virais/fisiologiaRESUMO
Hepatitis E virus- (HEV-) mediated hepatitis has become a global public health problem. An important regulatory protein of HEV, ORF3, influences multiple signal pathways in host cells. In this study, to investigate the function of ORF3 from the swine form of HEV (SHEV), high-throughput RNA-Seq-based screening was performed to identify the differentially expressed genes in ORF3-expressing HepG2 cells. The results were validated with quantitative real-time PCR and gene ontology was employed to assign differentially expressed genes to functional categories. The results indicated that, in the established ORF3-expressing HepG2 cells, the mRNA levels of CLDN6, YLPM1, APOC3, NLRP1, SCARA3, FGA, FGG, FGB, and FREM1 were upregulated, whereas the mRNA levels of SLC2A3, DKK1, BPIFB2, and PTGR1 were downregulated. The deregulated expression of CLDN6 and FREM1 might contribute to changes in integral membrane protein and basement membrane protein expression, expression changes for NLRP1 might affect the apoptosis of HepG2 cells, and the altered expression of APOC3, SCARA3, and DKK1 may affect lipid metabolism in HepG2 cells. In conclusion, ORF3 plays a functional role in virus-cell interactions by affecting the expression of integral membrane protein and basement membrane proteins and by altering the process of apoptosis and lipid metabolism in host cells. These findings provide important insight into the pathogenic mechanism of HEV.
Assuntos
Células Hep G2/virologia , Proteínas Virais/genética , Animais , Apoptose , Regulação da Expressão Gênica , Regulação Viral da Expressão Gênica , Vírus da Hepatite E , Humanos , Lentivirus/genética , Metabolismo dos Lipídeos , Proteínas de Membrana/química , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/química , Transdução de Sinais , Suínos , Transcriptoma , Regulação para CimaRESUMO
BACKGROUND: Hepatitis B virus (HBV) infection correlated with the development of cirrhosis, liver failure and hepatocellular carcinoma (HCC), poses a huge health burden on the global community. However, the pathogenesis of chronic hepatitis B (CHB) remains unclear. Apolipoprotein A1 (ApoA1) mainly secreted by hepatocytes, represents the major protein component of high-density lipoprotein. ApoA1 secretion may be disrupted by HBV infection. In this study, we mainly investigated the molecular mechanism of ApoA1 down regulated by HBV for revealing the pathogenesis of CHB. METHODS: ApoA1 expression in livers of CHB patients as well as healthy controls were performed by Real-time PCR (RT-PCR) and Western blot. The serum ApoA1 levels were measured by Enzymed-linked immunosorbent assay (ELISA). Expression of ApoA1 mRNA and protein levels were performed by RT-PCR and Western blot in human hepatoma HepG2 cells and subline HepG2.2.15 cells. HBV expression construct, pHBV1.3 were transfected into HepG2, the changes of ApoA1 mRNA and protein expression were detected by RT-PCR and Western blot. To further study the mechanism of ApoA1 down regulation by HBV, 11 CpG islands in ApoA1 promotor were tested for DNA methylation status by MSP. HepG2.2.15 cell lines were treated with DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-dC), then, expression of ApoA1 mRNA and HBV particles in the supernatant, as well as ApoA1 protein levels were detected by RT-PCR and Western blot. Secretion of HBsAg and HBeAg in HepG2 cells cotransfected with pApoA1 and pHBV1.3 constructs was tested by ELISA. Meanwhile, secretion of HBsAg and HBeAg in the supernatant were quantified by ELISA in the HepG2.2.15 cells treated with 5-aza-dC plus ApoA1 siRNA. RESULTS: Expression of ApoA1 mRNA and protein levels, as well as serum ApoA1 levels in CHB patients were decreased corresponding healthy controls in vivo. In addition, the expression of ApoA1 mRNA and protein levels were down regulated in HepG2.2.15 cells correponding HepG2 cells, 11 CpG islands in ApoA1 promoter were tested for methylation status by MSP in HepG2.2.15 cells compared to HepG2 cells, while two CpG islands were found hypermethylated. Expression of ApoA1 mRNA and protein levels were increased in HepG2.2.15 cells treated with DNA methyltransferase inhibitor 5-aza-dC. Furthermore, overexpression of ApoA1 can enhance HBV expression in HepG2 cells while the inhibitory effect of 5-aza-dC on HBV expression was completely abolished by blocking 5-aza-dC-induced up-regulation of ApoA1 using RNAi. CONCLUSIONS: Epigenetic silencing of ApoA1 gene expression by CpG island DNA hypermethylation induced by HBV may contribute to the pathogenesis of CHB.
Assuntos
Apolipoproteína A-I/genética , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/genética , Interações Hospedeiro-Patógeno , Apolipoproteína A-I/sangue , Apolipoproteína A-I/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Estudos de Casos e Controles , Ilhas de CpG , Metilação de DNA , Decitabina , Regulação para Baixo , Epigênese Genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2/efeitos dos fármacos , Células Hep G2/virologia , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/virologia , Humanos , Regiões Promotoras GenéticasRESUMO
Caveolae are flask-shaped invaginations of the plasma membrane. Caveolae play important roles in the process of viruses entry into host cells, but the roles of caveolae at the late stage of virus infection were not completely understood. Tiger frog virus (TFV) has been isolated from the diseased tadpoles of the frog, Rana tigrina rugulosa, and causes high mortality of tiger frog tadpoles cultured in Southern China. In the present study, the roles of caveolae at the late stage of TFV infection were investigated. We showed that TFV virions were localized with the caveolae at the late stage of infection in HepG2 cells. Disruption of caveolae by methyl-ß-cyclodextrin/nystatin or knockdown of caveolin-1 significantly increase the release of TFV. Moreover, the interaction between caveolin-1 and TFV major capsid protein was detected by co-immunoprecipitation. Those results suggested that caveolae restricted TFV release from the HepG2 cells. Caveolae-associated proteins (caveolin-1, caveolin-2, cavin-1, and cavin-2) were selectively incorporated into TFV virions. Different combinations of proteolytic and/or detergent treatments with virions showed that caveolae-associated proteins were located in viral capsid of TFV virons. Taken together, caveolae might be a restriction factor that affects virus release and caveolae-associated proteins were incorporated in TFV virions.
Assuntos
Cavéolas/fisiologia , Células Hep G2/virologia , Ranavirus/fisiologia , Liberação de Vírus , Proteínas do Capsídeo/metabolismo , Caveolina 1/metabolismo , Células Hep G2/metabolismo , Humanos , Vírion/fisiologia , Internalização do VírusRESUMO
Two new seco-prezizaane-type sesquiterpenes, 3,4-dehydroneomajucin (1) and 1,2,3,4-tetradehydroneomajucin (2), were isolated from the fruits of Illicium jiadifengpi. The structure of these compounds was determined using 1D and 2D NMR and ESI-MS. The isolates were evaluated for their anti-hepatitis B virus activities on the Hep G2.2.15 cell line. The inhibitory rates of compounds 1 and 2 on the HBeAg and HBsAg expression were 30.08 ± 3.09% and 11.43 ± 1.92% at a concentrations of 68.00 µM and 7.88 ± 1.21% and 16.96 ± 4.24% at a concentration of 68.50 µM, respectively.
Assuntos
Antivirais/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Illicium/química , Sesquiterpenos/química , Antivirais/química , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Frutas/química , Células Hep G2/efeitos dos fármacos , Células Hep G2/virologia , Humanos , Lactonas/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/farmacologia , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Anti-HBV therapy is essential for patients awaiting liver transplantation. This study aimed to explore the effects of dendritic cells (DCs) derived from the peripheral blood of hepatitis B patients on the replication of HBV in vivo and to evaluate the biosafety of DCs in clinical therapy. Peripheral blood mononuclear cells (PBMCs) were isolated from HBV-infected patients and maturation-promoting factors and both HBsAg and HBcAg were used to induce DC maturation. Mature DCs and lymphocytes were co-cultured with human hepatocyte cell HL-7702 or HBV-producing human hepatocellular carcinoma cell HepG2.2.15. We found that mature lymphocytes exposed to DCs in vitro did not influence morphology or activities of HL-7702 and HepG2.2.15 cells. Liver function indexes and endotoxin levels in the cell supernatants did not change in these co-cultures. Additionally, supernatant and intracellular HBV DNA levels were reduced when HepG2.2.15 cells were co-cultured with mature lymphocytes that had been cultured with DCs, and HBV covalently closed circular DNA (cccDNA) levels in HepG2.2.15 cells also decreased. Importantly, DC-mediated immunotherapy had no mutagenic effect on HBV genomic DNA by gene sequencing of the P, S, X, and C regions of HBV genomic DNA. We conclude that PBMC-derived DCs from HBV-infected patients act on autologous lymphocytes to suppress HBV replication and these DC clusters showed favorable biosafety.
Assuntos
Células Dendríticas/fisiologia , Vírus da Hepatite B/fisiologia , Leucócitos Mononucleares/virologia , Replicação Viral/fisiologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Técnicas de Cocultura , Meios de Cultivo Condicionados/análise , DNA Viral/análise , DNA Viral/genética , Células Dendríticas/virologia , Endotoxinas/análise , Células Hep G2/virologia , Hepatite B/sangue , Hepatite B/terapia , Vírus da Hepatite B/patogenicidade , Hepatócitos/fisiologia , Humanos , Ativação LinfocitáriaRESUMO
Hepatitis C virus (HCV) core protein stimulates many signaling pathways related to apoptosis inhibition resulting in hepatocellular carcinoma (HCC). It has been reported that sirt1 is involved in regulating apoptosis; therefore, we investigated the influence of HCV core protein on sirt1 expression and apoptosis in human HepG2 cells. Our study showed that HCV core protein inhibited apoptosis of HepG2 cells as well as caspase-3 expression and activity (P < 0.05). At the same time, sirt1 expression was increased at both the mRNA (P < 0.05) and protein (P < 0.05) levels. Furthermore, apoptosis inhibition was reversed when sirt1 was knocked down (P < 0.05). Our study provides further evidence that the sirt1-p53-Bax signaling pathway plays an important role in regulating the suppression of cell apoptosis induced by HCV core protein.
Assuntos
Apoptose/efeitos dos fármacos , Células Hep G2/virologia , Hepacivirus/fisiologia , Sirtuína 1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas do Core Viral/biossíntese , Proteínas do Core Viral/metabolismo , Proteína X Associada a bcl-2/metabolismo , Sequência de Bases , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/virologia , Técnicas de Silenciamento de Genes , Células Hep G2/citologia , Hepacivirus/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/virologia , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transdução de Sinais , Sirtuína 1/farmacocinética , Proteínas do Core Viral/genéticaRESUMO
Cs2K4Na[SiW9Nb3O40] has broad antiviral ability including anti-Influenza A, -Influenza B, -HSV-1, -HSV-2, -HIV-1, and -HBV. A series of antivirus and/or biochemical experiments and X-ray nanotomography analysis confirm that this kind of broad-spectrum antiviral property is mainly due to its localization on the cell surface.
Assuntos
Antivirais/química , Compostos de Tungstênio/química , Vírus/efeitos dos fármacos , Antivirais/farmacologia , Células Hep G2/química , Células Hep G2/virologia , Humanos , Propriedades de Superfície , Compostos de Tungstênio/farmacologiaRESUMO
RNA interference (RNAi) is a powerful gene knockdown technique used for study gene function. It also potentially provides effective agents for inhibiting infectious and genetic diseases. Most of RNAi studies employ a single siRNA designing program and then require large-scale screening experiments to identify functional siRNAs. In this study, we demonstrate that an assembly of results generated from different siRNA designing programs could provide clusters of predicting sites that aided selection of potent siRNAs. Based on the clusters, three siRNA target sites were selected on a conserved RNA region of hepatitis B virus (HBV), known as HBV post-transcriptional regulatory element (HBV PRE) at nucleotide positions 1317-1337, 1357-1377 and 1644-1664. All three chosen siRNAs driven by H1 promoter were highly effective and could drastically decrease expression of HBV transcripts (core, surface and X) and surface protein without induction of interferon response and cell cytotoxicity in liver cancer cell line (HepG2). Based on prediction of secondary structures, the silencing effects of siRNAs were less effective against a loop sequence of the mRNA target with hairpin structure. In summary, we demonstrate an effectual approach for identification of functional siRNAs. Moreover, highly potent siRNAs identified here may serve as novel agents for development of nucleic acid-based HBV therapy.
Assuntos
Vírus da Hepatite B/genética , RNA Interferente Pequeno/genética , 2',5'-Oligoadenilato Sintetase/genética , Algoritmos , Sequência de Bases , Regulação da Expressão Gênica , Células Hep G2/virologia , Humanos , Luciferases/genética , Luciferases/metabolismo , Dados de Sequência Molecular , NF-kappa B/genética , Regiões Promotoras Genéticas , RNA Interferente Pequeno/química , Sequências Reguladoras de Ácido Nucleico , Fator de Transcrição STAT1/genética , TermodinâmicaRESUMO
UNLABELLED: Hepatitis C virus (HCV) exposure leads to persistent life-long infections characterized by chronic inflammation often developing into cirrhosis and hepatocellular carcinoma. The mechanism by which HCV remains in the liver while inducing an inflammatory and antiviral response remains unclear. Though the innate immune response to HCV in patients seems to be quite active, HCV has been shown in cell culture to employ a diverse array of innate immune antagonists, which suggests that current model systems to study interactions between HCV and the innate immune system are not representative of what happens in vivo. We recently showed that hepatoma-derived HepG2 cells support the entire HCV life cycle if the liver-specific microRNA, miR-122, is expressed along with the entry factor, CD81 (termed HepG2-HFL cells). We found that there was a striking difference in these cells' ability to sustain HCV infection and spread when compared with Huh-7 and Huh-7.5 cells. Additionally, HepG2-HFL cells exhibited a more robust antiviral response when challenged with other RNA viruses and viral mimetics than Huh-7 and Huh-7.5 cells. HCV infection elicited a potent interferon-lambda (IFN-λ), IFN-stimulated gene, and cytokine response in HepG2-HFL cells, but not in Huh-7 cells, suggesting that HepG2-HFL cells more faithfully recapitulate the innate immune response to HCV infection in vivo. Using this model, we found that blocking the retinoic acid-inducible gene I (RIG-I)-like receptor pathway or the IFN-λ-signaling pathway promoted HCV infection and spread in HepG2-HFL cells. CONCLUSION: HepG2-HFL cells represent a new system to study the interaction between HCV and the innate immune system, solidifying the importance of IFN-λ in hepatic response to HCV infection and revealing non-redundant roles of RIG-I and melanoma differentiation-associated protein 5 in HCV recognition and repression of infection.
Assuntos
Células Hep G2/metabolismo , Células Hep G2/virologia , Hepacivirus/fisiologia , Hepatite C/metabolismo , Hepatite C/fisiopatologia , Imunidade Inata/fisiologia , Interleucinas/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Citocinas/metabolismo , Proteína DEAD-box 58 , RNA Helicases DEAD-box/metabolismo , Células Hep G2/patologia , Hepatite C/patologia , Humanos , Helicase IFIH1 Induzida por Interferon , Interferons , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , MicroRNAs/metabolismo , Receptores Imunológicos , Transdução de Sinais/fisiologia , Replicação Viral/fisiologiaRESUMO
Hepatitis B surface antigen (HBsAg) seropositivity is an important risk factor for hepatocellular carcinoma (HCC), and HBsAg-transgenic mice have been reported to spontaneously develop HCC. The major histocompatibility complex class I-related molecules A and B (MICA and MICB) are NKG2D ligands that play important roles in tumor immune surveillance. In the present study, we found that HBsAg overexpression in HepG2 cells led to upregulation of 133 and downregulation of 9 microRNAs (miRNAs). Interestingly, several HBsAg-induced miRNAs repressed the expression of MICA and MICB via targeting their 3'-untranslated regions. In addition, the expression of MICA and MICB was significantly reduced upon HBsAg overexpression, which was partially restored by inhibiting the activities of HBsAg-induced miRNAs. Moreover, HBsAg-overexpressing HCC cells exhibited reduced sensitivity to natural killer cell-mediated cytolysis. Taken together, our data suggest that HBsAg supresses the expression of MICA and MICB via induction of cellular miRNAs, thereby preventing NKG2D-mediated elimination of HCC cells.
Assuntos
Carcinoma Hepatocelular/virologia , Antígenos de Superfície da Hepatite B/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Neoplasias Hepáticas/virologia , MicroRNAs/genética , Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Células Hep G2/virologia , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Vírus da Hepatite B/fisiologia , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Neoplasias Hepáticas/genéticaRESUMO
Viral infections of host cells cause multiple changes of cellular metabolism including immediate defense mechanisms as well as processes to support viral replication. Coxsackievirus B3 (CVB3) is a member of the Picornavirus family and is responsible for a wide variety of mild or severe infections including acute and chronic inflammations. Thereby, intracellular signaling can be changed very comprehensively. In order to compare the influence of CVB3 replication on gene expression pattern of two different cell lines, DNA microarray systems were used to study a set of 780 genes related to inflammation. Expression analysis of HeLa cells and HepG2 cells infected with CVB3 identified 34 genes whose mRNA levels were altered significantly upon infection. The expression of additional 16 genes in HepG2 cells and 31 genes in HeLa cells were found to be influenced during CVB3 replication as well. All genes expressed differentially were sorted with regard to their functions and interpreted in view of known contributors to the infection process. The activation of the tumor necrosis factor pathways by CVB3 represents one peculiar observation, including apoptosis, stress, and inflammation responses.
Assuntos
Infecções por Coxsackievirus/genética , Infecções por Coxsackievirus/metabolismo , Enterovirus Humano B/genética , RNA Mensageiro/biossíntese , Apoptose/genética , Linhagem Celular Tumoral , Infecções por Coxsackievirus/virologia , Ativação Enzimática , Expressão Gênica , Perfilação da Expressão Gênica , Células HeLa/virologia , Células Hep G2/virologia , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Replicação Viral/genéticaRESUMO
Hepatitis B virus (HBV) is the most common of the hepatitis viruses that cause chronic liver infections in humans and it is considered a major global health problem. However, the mechanisms of HBV replication are complex and not yet fully understood. In this study, the HBV DNA-transfected HepG2.2.15 cell line and its parental HepG2 cell line were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ)-coupled two-dimensional liquid chromatography tandem mass-spectrophotometry (2D LC-MS/MS), a successfully exploited high-throughput proteomic technology. In total, 2,028 unique proteins were identified and 170 proteins were differentially expressed in HepG2.2.15 cells as compared with that in HepG2. Several differentially expressed proteins were further validated by Western blot and real-time quantitative reverse transcription-PCR. Furthermore, the association of HBV replication with heat shock protein B1, one of the highly expressed proteins in HepG2.2.15 cells, was verified. HSPB1 functions as a anti-viral protein during HBV infection by specifically inducing type interferon and some downstream antiviral effectors. This study is the first to report the application of iTRAQ technology to analyze the underlying mechanisms of HBV replication. Many of the differentially expressed proteins identified have not been linked to HBV replication before, and may provide valuable novel insights into HBV replication.