RESUMO
BACKGROUND: Chronic hepatitis B virus (HBV) infection is an important cause of cirrhosis and hepatocellular carcinoma. The major challenges for current therapies are the low efficacy of current drugs and the occurrence of drug resistant HBV mutations. RNA interference (RNAi) of virus-specific genes offers the possibility of developing a new anti-HBV therapy. Recent reports have shown that lentiviral vectors based on HIV-1 are promising gene delivery vehicles due to their ability to integrate transgenes into non-dividing cells. Herein, a lentivirus-based RNAi system was developed to drive expression and delivery of HBV-specific short hairpin RNA (shRNA) in a mouse model for HBV replication. METHODS: Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in the sera of the mice were analyzed by quantitative sandwich enzyme linked immunosorbent assay (ELISA) technique, hepatitis B core antigen (HBcAg) and HBsAg in the livers of the mice were detected by immunohistochemical assay, HBV DNA and HBV mRNA were measured by fluorogenic quantitative polymerase chain reaction (FQ-PCR) and quantitative real-time PCR respectively. RESULTS: Co-injection of HBV plasmids together with the lentivirus targeting HBV shRNA induced an RNAi response. Secreted HBsAg was reduced by 89% in mouse serum, and HBeAg was also significantly inhibited, immunohistochemical detection of HBcAg or HBsAg in the liver tissues also revealed substantial reduction. Lentiviral mediated shRNA caused a significant suppression in the levels of viral mRNA and DNA synthesis compared to the control group. CONCLUSION: Lentivirus-based RNAi can be used to suppress HBV replication in vivo, it might become a potential therapeutic strategy for treating HBV and other viral infections.
Assuntos
Vetores Genéticos/uso terapêutico , Vírus da Hepatite B/fisiologia , Lentivirus/genética , Interferência de RNA/fisiologia , Replicação Viral/fisiologia , Animais , DNA Viral/genética , Modelos Animais de Doenças , Feminino , Vetores Genéticos/genética , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Antígenos de Superfície da Hepatite B/metabolismo , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Hepatite B Crônica/terapia , Fígado/metabolismo , Fígado/virologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , RNA Viral/genética , Replicação Viral/genéticaRESUMO
OBJECTIVE: To study the efficacy of anti-telomerase siRNA in hepatocellular carcinoma both in vitro and in vivo. METHODS: Lentvirus vectors contained anti-telomerase siRNA were conducted with a high performance homologous recombination system, and then were transduced into human hepatocellular carcinoma HepG2 cells. The telomerase activity was detected by RT-PCR, HepG2 cell proliferation was determined by MTT assay, and apoptosis was detected by TUNEL assay. The in vivo experiment was carried out by inoculation of HepG2 cells into nude mice and the tumor growth was measured and analyzed. RESULTS: The growth of transfected HepG2 cells was significantly inhibited and the inhibition rate was 57.5% at the 8th day after transfection. The telomerase activity was significantly suppressed in vitro. The growth of transfected human hepatocellular HepG2 tumor in the nude mice was also significantly inhibited. CONCLUSION: The results of this study demonstrate that the growth of hepatocellular carcinoma cells is effectively inhibited by transfection of anti-telomerase siRNA both in vitro and in vivo.