Summary.
In this study, the effect of RNAi on HBV replication was observed in a cell culture model, HepG2 2.2.15 cell line, which supports human HBV ayw replication and expression. Aim of the study was to investigate effects of shRNAs (small hairpin RNAs) targeting hepatitis B virus mRNAs on the viral replication in HepG2 2.2.15 cells. We selected three target HBV mRNA regions with different putative secondary structures to test whether the secondary structure of RNA may affect the inhibition efficacy on the target HBV RNA. Three HBV-specific siRNAs (small interfering RNA) were designed targeting X (1689–1708), Core (2229–2248) and S (765–784 nt) transcripts. HepG2 2.2.15 cells were transfected with shRNA expressing plasmids, P765, P2229 and P1689 targeting S, core and X region, respectively or a mock plasmid targeting lacZ gene. The culture media was collected throughout six days after transfection and analyzed by real-time PCR. Viral DNA production was suppressed for 7 days. The HBV DNA levels were decreased by 73, 72 and 79% with P765, P2229 and P1689 vectors, respectively.
In conclusion, the shRNAs designed for X, core and S regions, specifically and significantly suppressed HBV DNA. siRNAs potentially may be used in treatment of hepatitis B.
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References
P Arbuthnot S Carmona A Ely (2005) ArticleTitleExploiting the RNA interference pathway to counter HBV replication Liver Int 25 9–15 10.1111/j.1478-3231.2004.0966.x Occurrence Handle10.1111/j.1478-3231.2004.0966.x Occurrence Handle1:CAS:528:DC%2BD2MXisFCqtrc%3D Occurrence Handle15698393
EA Bohula AJ Salisbury M Sohail MP Playford J Riedemann EM Southern VM Macaulay (2003) ArticleTitleThe efficacy of small interfering RNAs targeted to the type 1 insulin-like growth factor receptor (IGF1R) is influenced by secondary structure in the IGF1R transcript J Biol Chem 278 15991–15997 10.1074/jbc.M300714200 Occurrence Handle10.1074/jbc.M300714200 Occurrence Handle1:CAS:528:DC%2BD3sXjt1ehsb0%3D Occurrence Handle12604614
Chen CC, Ko TM, Ma HI, Wu HL, Xiao X, Li J, Chang CM, Wu PY, Chen CH, Han JM, Yu CP, Jeng KS, Hu CP, Tao MH (2006) Long-term inhibition of hepatitis B virus in transgenic mice by double-stranded adeno-associated virus 8-delivered short hairpin RNA. Gene Ther Aug 24
Y Chen D Du J Wu CP Chan Y Tan HF Kung ML He (2003) ArticleTitleInhibition of hepatitis B virus replication by stably expressed shRNA Biochem Biophys Res Commun 311 398–404 10.1016/j.bbrc.2003.10.009 Occurrence Handle10.1016/j.bbrc.2003.10.009 Occurrence Handle1:CAS:528:DC%2BD3sXosFSntb8%3D Occurrence Handle14592428
Z Chen ZF Xu J Ye H Yao S Zheng J Ding (2005) ArticleTitleCombination of small interfering RNAs mediates greater inhibition of human hepatitis B virus replication and antigen expression J Zhejiang Univ Sci 68 236–241 10.1631/jzus.2005.B0236 Occurrence Handle10.1631/jzus.2005.B0236
TL Cheng WW Chang IJ Su MD Lai W Huang HY Lei WT Chang (2005) ArticleTitleTherapeutic inhibition of hepatitis B virus surface antigen expression by RNA interference Biochem Biophys Res Commun 336 820–830 10.1016/j.bbrc.2005.08.173 Occurrence Handle10.1016/j.bbrc.2005.08.173 Occurrence Handle1:CAS:528:DC%2BD2MXhtVegtb7J Occurrence Handle16153600
HS Conjeevaram AS Lok (2003) ArticleTitleManagement of chronic hepatitis B J Hepatol 38 S90–S103 10.1016/S0168-8278(02)00431-2 Occurrence Handle10.1016/S0168-8278(02)00431-2 Occurrence Handle12591188
DM Dykxhoorn J Lieberman (2006) ArticleTitleRunning interference: prospects and obstacles to using small interfering RNAs as small molecule drugs Annu Rev Biomed Eng 8 377–402 10.1146/annurev.bioeng.8.061505.095848 Occurrence Handle10.1146/annurev.bioeng.8.061505.095848 Occurrence Handle1:CAS:528:DC%2BD28Xpt1CltLY%3D Occurrence Handle16834561
DM Dykxhoorn CD Novina PA Sharp (2003) ArticleTitleKilling the messenger: short RNAs that silence gene expression Nat Rev Mol Cell Biol 4 457–467 10.1038/nrm1129 Occurrence Handle10.1038/nrm1129 Occurrence Handle1:CAS:528:DC%2BD3sXktFOns74%3D Occurrence Handle12778125
SM Elbashir J Harborth K Weber T Tuschl (2002) ArticleTitleAnalysis of gene function in somatic mammalian cells using small interfering RNAs Methods 26 199–213 10.1016/S1046-2023(02)00023-3 Occurrence Handle10.1016/S1046-2023(02)00023-3 Occurrence Handle1:CAS:528:DC%2BD38Xks1GhtLw%3D Occurrence Handle12054897
SM Elbashir W Lendeckel T Tuschl (2001) ArticleTitleRNA interference is mediated by 21- and 22-nucleotide RNAs Genes Dev 15 188–200 10.1101/gad.862301 Occurrence Handle10.1101/gad.862301 Occurrence Handle1:CAS:528:DC%2BD3MXhvVOjtbg%3D Occurrence Handle11157775
DA Gadkari (2005) ArticleTitleRNA interference and inhibition of viruses Indian J Med Res 121 147–150 Occurrence Handle1:CAS:528:DC%2BD2MXjvVyltrg%3D Occurrence Handle15802755
H Giladi M Ketzinel-Gilad L Rivkin Y Felig O Nussbaum E Galun (2003) ArticleTitleSmall interfering RNA inhibits hepatitis B virus replication in mice Mol Ther 8 769–776 10.1016/S1525-0016(03)00244-2 Occurrence Handle10.1016/S1525-0016(03)00244-2 Occurrence Handle1:CAS:528:DC%2BD3sXos1Cgsr8%3D Occurrence Handle14599810
K Hamasaki K Nakao K Matsumoto T Ichikawa H Ishikawa K Eguchi (2003) ArticleTitleShort interfering RNA-directed inhibition of hepatitis B virus replication FEBS Lett 543 51–54 10.1016/S0014-5793(03)00400-9 Occurrence Handle10.1016/S0014-5793(03)00400-9 Occurrence Handle1:CAS:528:DC%2BD3sXjs1Crsbw%3D Occurrence Handle12753904
GJ Hannon (2002) ArticleTitleRNA interference Nature 418 244–251 10.1038/418244a Occurrence Handle10.1038/418244a Occurrence Handle1:CAS:528:DC%2BD38XltFGmu7Y%3D Occurrence Handle12110901
F Jia YZ Zhang CM Liu (2006) ArticleTitleA retrovirus-based system to stably silence hepatitis B virus genes by RNA interference Biotechnol Lett 28 1679–1685 10.1007/s10529-006-9138-z Occurrence Handle10.1007/s10529-006-9138-z Occurrence Handle1:CAS:528:DC%2BD28XptF2rs78%3D Occurrence Handle16900331
C Klein CT Bock H Wedemeyer T Wustefeld S Locarinini HP Dienes S Kubicka MP Manns C Trautwein (2003) ArticleTitleInhibition of hepatitis B virus replication in vivo by nucleoside analogues and siRNA Gastroenterology 125 9–18 10.1016/S0016-5085(03)00720-0 Occurrence Handle10.1016/S0016-5085(03)00720-0 Occurrence Handle1:CAS:528:DC%2BD3sXmtVWqtb0%3D Occurrence Handle12851866
M Konishi CH Wu GY Wu (2003) ArticleTitleInhibition of HBV replication by siRNA in a stable HBV-producing cell line Hepatology 38 842–850 Occurrence Handle1:CAS:528:DC%2BD3sXovFGhtb8%3D Occurrence Handle14512871
HS Margolis (1998) ArticleTitleHepatitis B virus infection Bull WHO 76 152–153 Occurrence Handle10063701
N Moolla M Kew P Arbuthnot (2002) ArticleTitleRegulatory elements of hepatitis B virus transcription J Viral Hepat 9 323–331 10.1046/j.1365-2893.2002.00381.x Occurrence Handle10.1046/j.1365-2893.2002.00381.x Occurrence Handle12225325
MD Moore MJ McGarvey RA Russell B Cullen MO McClure (2005) ArticleTitleStable inhibition of hepatitis B virus proteins by small interfering RNA expressed from viral vectors J Gene Med 7 918–925 10.1002/jgm.739 Occurrence Handle10.1002/jgm.739 Occurrence Handle1:CAS:528:DC%2BD2MXos1aqsrk%3D Occurrence Handle15756649
P Provost D Dishart J Doucet D Frendewey B Samuelsson O Radmark (2002) ArticleTitleRibonuclease activity and RNA binding of recombinant human Dicer EMBO J 21 5864–5874 10.1093/emboj/cdf578 Occurrence Handle10.1093/emboj/cdf578 Occurrence Handle1:CAS:528:DC%2BD38XosVSrtLk%3D Occurrence Handle12411504
X Ren G Luo Z Xie L Zhou X Kong A Xu (2006) ArticleTitleInhibition of multiple gene expression and virus replication of HBV by stable RNA interference in 2.2.15 cells J Hepatology 44 663–670 10.1016/j.jhep.2005.10.029 Occurrence Handle10.1016/j.jhep.2005.10.029 Occurrence Handle1:CAS:528:DC%2BD28XisFyqt7s%3D
XR Ren LJ Zhou GB Luo A Xu (2005) ArticleTitleInhibition of hepatitis B virus replication in 2.2.15 cells by expressed shRNA J Viral Hepat 12 236–242 10.1111/j.1365-2893.2005.00587.x Occurrence Handle10.1111/j.1365-2893.2005.00587.x Occurrence Handle15850463
MA Sells ML Chen G Acs (1987) ArticleTitleProduction of hepatitis B virus particles in HepG2 cells transfected with cloned hepatitis B virus DNA Proc Natl Acad Sci USA 84 1005–1009 10.1073/pnas.84.4.1005 Occurrence Handle10.1073/pnas.84.4.1005 Occurrence Handle1:CAS:528:DyaL2sXhvV2qt7c%3D Occurrence Handle3029758
A Shlomai Y Shaul (2003) ArticleTitleInhibition of hepatitis B virus expression and replication by RNA interference Hepatology 37 764–770 10.1053/jhep.2003.50146 Occurrence Handle10.1053/jhep.2003.50146 Occurrence Handle1:CAS:528:DC%2BD3sXjt1Wltbs%3D Occurrence Handle12668968
M Singh A Dicaire AE Wakil C Luscombe SL Sacks (2004) ArticleTitleQuantitation of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in the liver of HBV-infected patients by LightCyclerTM real-time PCR J Virol Methods 118 159–167 10.1016/j.jviromet.2004.02.006 Occurrence Handle10.1016/j.jviromet.2004.02.006 Occurrence Handle1:CAS:528:DC%2BD2cXjtFaltbg%3D Occurrence Handle15081611
SL Uprichard B Boyd A Althage FV Chisari (2005) ArticleTitleClearance of hepatitis B virus from the liver of transgenic mice by short hairpin RNAs Proc Natl Acad Sci USA 102 773–778 10.1073/pnas.0409028102 Occurrence Handle10.1073/pnas.0409028102 Occurrence Handle1:CAS:528:DC%2BD2MXis1CqsLY%3D Occurrence Handle15640346
PM Waterhouse MB Wang T Lough (2001) ArticleTitleGene silencing as an adaptive defense against viruses Nature 411 834–842 10.1038/35081168 Occurrence Handle10.1038/35081168 Occurrence Handle1:CAS:528:DC%2BD3MXksF2gu7Y%3D Occurrence Handle11459066
HL Wu LR Huang CC Huang HL Lai CJ Liu YT Huang YW Hsu CY Lu DS Chen (2005) ArticleTitleRNA interference-mediated control of hepatitis B virus and emergence of resistant mutant Gastroenterology 128 708–716 10.1053/j.gastro.2004.12.007 Occurrence Handle10.1053/j.gastro.2004.12.007 Occurrence Handle1:CAS:528:DC%2BD2MXivFWntLY%3D Occurrence Handle15765406
C Ying E De Clercq J Neyts (2003) ArticleTitleSelective inhibition of hepatitis B virus replication by RNA interference Biochem Biophys Res Commun 309 482–484 10.1016/j.bbrc.2003.08.021 Occurrence Handle10.1016/j.bbrc.2003.08.021 Occurrence Handle1:CAS:528:DC%2BD3sXmvVKmsrg%3D Occurrence Handle12951075
Ying RS, Zhu C, Fan XG, Li N, Tian XF, Liu HB, Zhang BX (2006) Hepatitis B virus is inhibited by RNA interference in cell culture and in mice. Antivir Res Jun 28
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Kayhan, H., Karatayli, E., Turkyilmaz, A. et al. Inhibition of hepatitis B virus replication by shRNAs in stably HBV expressed HEPG2 2.2.15 cell lines. Arch Virol 152, 871–879 (2007). https://doi.org/10.1007/s00705-006-0918-5
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DOI: https://doi.org/10.1007/s00705-006-0918-5