RESUMO
BACKGROUND: Chronic infection with hepatitis B virus (HBV) is widespread because of the limited availability of therapeutic treatments. Although previous reports have suggested that RNA interference has promise as a treatment for HBV infection, further studies of long-term and off-target drug effects on HBV, especially on drug-resistant strains of HBV, are needed. Therefore, seven vectors that express short hairpin RNAs (shRNAs), driven by the polymerase II promoter, pSilencer4.1/HBV, were constructed to target open reading frames (ORFs) of the HBV C and S genes from wild-type and drug-resistant strains. Treatment efficiency was also assessed. METHODS: The pSilencer4.1/HBV vectors were investigated in HepG2.2.15 cells and transgenic mice that consistently produce wild-type HBV. Additionally, vectors that produce a lamivudine-resistant strain of HBV were developed and cotransfected, along with pSilencer/HBV, into both HepG2 cells and mice. The effects of polymerase-II-driven pSilencer4.1/HBV were compared with those of polymerase-III-driven pSilencer3.1/HBV at both the gene and protein level. RESULTS: pSilencer4.1/HBV inhibited the expression of viral protein, DNA and HBV subtype ayw mRNA in both HepG2.2.15 cells and transgenic mice. Toxicity, as well as off-target effects, did not occur after a short- to medium-term examination. Moreover, an HBV strain resistant to lamivudine, subtype adr, was suppressed by shRNA in both HepG2 cells and mice. In contrast to polymerase III, vectors that used polymerase II could drive efficient silencing without off-target effects. CONCLUSIONS: Silencing by shRNA dramatically inhibited HBV expression and replication regardless of strain type. ShRNA could therefore be a promising treatment for HBV infection.
Assuntos
Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/terapia , Lamivudina/farmacologia , Interferência de RNA , RNA Interferente Pequeno/uso terapêutico , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Linhagem Celular Tumoral , DNA Viral , Farmacorresistência Viral , Regulação Viral da Expressão Gênica , Vetores Genéticos , Antígenos de Superfície da Hepatite B/isolamento & purificação , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/metabolismo , Humanos , Lamivudina/uso terapêutico , Fígado/citologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , RNA Polimerase II/metabolismo , RNA MensageiroRESUMO
Toll-like receptor 4 (TLR4) is critical for activation of macrophages by Lipopolysaccharide (LPS). In this study, we investigated the silencing effects of TLR4-specific 21-nt small interfering RNAs (siRNA) on TLR4 expression in RAW264.7 cells. It was found that treatment with TLR4 siRNA down-regulated the TLR4 mRNA and protein expression in macrophage RAW264.7 cells, and reduced the sensitivity of the cells to LPS stimulation. Our findings also demonstrate that treatment with TLR4 siRNA significantly decreased the tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein 2 (MIP-2) expression induced by LPS. TLR4 siRNA treatment also impaired the signalling of mitogen-activated protein kinases (MAPK) induced by LPS in RAW264.7 cells. These data suggest that inhibition of TLR4 expression by TLR4 siRNA may be therapeutically beneficial in controlling the overall responses of immune cells to LPS.
Assuntos
Citocinas/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Animais , Linhagem Celular , Quimiocinas/metabolismo , Inativação Gênica , Macrófagos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Receptor 4 Toll-Like/metabolismo , TransfecçãoRESUMO
RNA interference might be an efficient antiviral therapy for some obstinate illness. Here, we studied the effects of hepatitis B virus (HBV)-specific 21-nt small interfering RNAs (siRNA) on HBV gene expression and replication in 2.2.15 cells. Seven vectors expressing specific hairpin siRNA driven by the RNA polymerase II-promoter were constructed and transfected into 2.2.15 cells. In the cell strain that can stably express functional siRNA, the HBV surface antigen (HBsAg) and the HBV e antigen (HBeAg) secretion into culture media was inhibited by 86% and 91%, respectively, as shown by an enzyme-linked immunosorbent assay. Immunofluorescence and Western blot indicated similar results. HBV DNA was markedly restrained by 3.28-fold, as assessed by the fluorescent quantitation PCR. Moreover, the HBV mRNA was significantly reduced by 80% based on semiquantitative RT-PCR. In conclusion, the specific siRNA can knock down the HBV gene expression and replication in vitro, and the silence effects have no relationship with interferon response.