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Inhibition of HBV replication by siRNA in a stable HBV‐producing cell line
Author(s) -
Konishi Masayoshi,
Wu Catherine H.,
Wu George Y.
Publication year - 2003
Publication title -
hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.488
H-Index - 361
eISSN - 1527-3350
pISSN - 0270-9139
DOI - 10.1002/hep.1840380410
Subject(s) - small interfering rna , microbiology and biotechnology , hepatitis b virus , hbsag , transfection , rna silencing , gene silencing , biology , rna interference , rna , virology , messenger rna , viral replication , cell culture , gene , virus , genetics , biochemistry
Potent inhibition of endogenous gene expression by RNA interference has been achieved by using sequence‐specific posttranscriptional gene silencing through the action of small interfering RNA molecules (siRNA). In these reports, the natural function of genes could be deduced through the ensuing loss of function. Based on the extraordinary effectiveness in silencing endogenous genes, we wondered whether siRNA could be applied against viral replication in a hepatitis B virus (HBV) model using HBV‐specific siRNA. To test this idea, HepG2 2.2.15, a human hepatoblastoma cell line that constitutively produces infectious HBV particles, was transfected with HBV‐specific siRNAs and controls. HBV surface antigen (HBsAg) secretion into culture media was inhibited by 78%, 67%, and 42% with siRNA against the polyadenylation (PA), precore (PreC), and surface (S) regions, respectively, compared with controls as detected by enzyme‐linked immunosorbent assay. After exposure to HBVPA siRNA, Northern blot analysis showed that HBV pregenomic RNA levels were decreased by 72%, and levels of HBV RNA containing the polyadenylation signal sequence were suppressed by 86%, as detected by RNase protection assay. Levels of HBV coreassociated DNA, a replication intermediate, also decreased by 71%. Immunocytochemistry revealed that 30% to 40% of the cells transfected with HBVPA siRNA were completely negative for detectable HBsAg levels. Controls consisting of treatment with HBV‐specific siRNA alone, lipofection reagent alone, or random double‐stranded RNA (dsRNA) lipofection complex failed to decrease HBV surface antigen, HBV messenger RNA (mRNA), or core‐associated HBV‐DNA levels. In conclusion, siRNA inhibits hepatitis B viral replication in a cell culture system. Future studies are needed to explore the specific delivery of siRNA to liver cells in vivo and the applicability of this approach. (Hepatology 2003;38:842–850).