Abstract
Objectives: To evaluate the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of hepatitis B virus (HBV) transcripts on the viral replication and antigen expression in vitro. Methods: (1) Seven siRNAs targeting surface (S), polymerase (P) or precore (PreC) region of HBV genome were designed and chemically synthesized. (2) HBV-producing HepG2.2. 15 cells were treated with or without siRNAs for 72 h. (3) HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. (4) Intracellular viral DNA was quantified by real-time PCR (Polymerase Chain Reaction). (5) HBV viral mRNA was reverse transcribed and quantified by real-time PCR. (6) The change of cell cycle and apoptosis was determined by flow cytometry. Results: Our data demonstrated that synthetic small interfering RNAs (siRNAs) targeting S and PreC gene could efficiently and specifically inhibit HBV replication and antigen expression. The expression of HBsAg and HBeAg and the replication of HBV could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, our results showed that the combination of siRNAs targeting various regions could inhibit HBV replication and antigen expression in a more efficient way than the use of single siRNA at the same final concentration. No apoptotic change was observed in the cell after siRNA treatment. Conclusion: Our results demonstrated that siRNAs exerted robust and specific inhibition on HBV replication and antigen expression in a cell culture system and combination of siRNAs targeting different regions exhibited more potency.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Reference
Beverly, L., Davidson, P.D., 2003. Hepatic diseases-hitting the target with inhibitory RNAs.N Engl J Med,349(24): 2357–2359.
Brummelkamp, T.R., Bernards, R., Agami, R., 2002. A system for stable expression of short interfering RNAs in mammalian cells.Science,296:550–553.
Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., Tuschl, T., 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature,411(6836):494–498.
Fung, S.K., Lok, A.S., 2004. Management of hepatitis B patients with antiviral resistance.Antivir Ther,9(6): 1013–1026.
Hamasaki, K., Nakao, K., Matsumoto, K., Ichikawa, T., Ishikawa, H., Eguchi, K., 2003. Short interfering RNA-directed inhibition of hepatitis B virus replication.FEBS Lett,543(1–3):51–54.
Hammond, S.M., Boettcher, S., Caudy, A.A., Kobayashi, R., Hannon, G.J., 2001. Argonaute2, a link between genetic and biochemical analyses of RNAi.Science,293(5532): 1146–1150.
Hannon, G.J., 2002. RNA interference.Nature,418(6894): 244–251.
Hilla, G., Mali, K.G., Ludmila, R., Yaakov, F., Ofer, N., Eithan, G.S., 2003. Small interfering RNA Inhibits Hepatitis B virus replication in mice.Molecular Therapy,8(5): 769–776.
Jacque, J.M., Triques, K., Stevenson, M., 2002. Modulation of HIV-1 replication by RNA interference.Nature,418(6896):435–438.
Kao, J.H., Chen, D.S., 2002. Global control of hepatitis B virus infection.Lancet Infect Dis,2(7):395–403.
Kapadia, S.B., Brideau-Andersen, A., Chisari, F.V., 2003. Interference of hepatitis C virus RNA replication by short interfering RNAs.Proc Natl Acad Sci USA,100(4): 2014–2018.
Lavanchy, D., 2004. Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures.J Viral Hepat,11(2):1145–1153.
Lee, W.M., 1997. Hepatitis B virus infection.N Engl J Med,337(24):1733–1745.
Liu, C.M., Liu, D.P., Dong, W.J., Liang, C.C., 2004. Retrovirus vector-mediated stable gene silencing in human cell.Biochem Biophys Res Commun,313(3):716–720.
Masayoshi, K., Catherine, H.W., George, Y.W., 2003. Inhibition of HBV replication by siRNA in a stable HBV-producing cell line.Hepatology,38(4):842–850.
McCaffrey, A.P., Nakai, H., Pandey, K., Huang, Z., Salazar, F.H., Xu, H., Wieland, S.F., Marion, P.L., Kay, M.A., 2003. Inhibition of hepatitis B virus in mice by RNA interference.Nat Biotechnol,21(6):639–644.
Plasterk, R.H.A., 2002. RNA silencing: the genome's immune system.Science,296(5571):1263–1265.
Sells, M.A., Chen, M.L., Acs, G., 1987. Production of hepatitis B particles in Hep G2 cells transfected with cloned hepatitis B virus DNA.Proc Natl Acad Sci USA,84(4): 1005–1009.
Shlomai, A., Shaul, Y., 2003. Inhibition of hepatitis B virus expression and replication by RNA interference.Hepatology,37(4):764–770.
Tatsuo, K., Yuri, K., Osamu, Y., Verena, G.M., 2004. Interference of hepatitis A virus replication by small interfering RNAs.Biochemical and Biophysical Research Communications,318(2):341–345.
Turelli, P., Mangeat, B., Jost, S., Vianin, S., Trono, D., 2004. Inhibition of hepatitis B virus replication by APOBEC3G.Science,303(5665):1829.
Ying, C.X., Erik, D.C., Johan, N., 2003. Selective inhibition of hepatitis B virus replication by RNA interference.Biochemical and Biophysical Research Communications,309(2):482–484.
Zamore, P.D., 2002. Ancient pathways programmed by small RNAs.Science,296(5571):1265–1269.
Zhao, L.J., Jian, H., Zhu, H., 2003. Specific gene inhibition by adenovirus-mediated expression of small interfering RNA.Gene,316:137–141.
Author information
Authors and Affiliations
Additional information
Project (No. 30471943) supported partly by the National Natural Science Foundation of China
Rights and permissions
About this article
Cite this article
Zhe, C., Ze-feng, X., Jing-jia, Y. et al. Combination of small interfering RNAs mediates greater inhibition of human hepatitis B virus replication and antigen expression. J Zheijang Univ Sci B 6, 236–241 (2005). https://doi.org/10.1007/BF02842458
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02842458