title: Study of the efficacy of combination therapy of SiRNAs in HepG2.2.15 cells.
abstract: OBJECTIVE
  Hepatitis B virus (HBV) infection is a world-wide health problem. The major obstacles for current anti-HBV therapy are the low efficacy and the occurrence of drug resistant HBV mutations. Recent studies have demonstrated that combination therapy can enhance antiviral efficacy and overcome the shortcomings. Here, the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of HBV nuclear localization signal (NLS) was monitored in HepG2.2.15 cells.
  
  
  METHODS
  Recombinant plasmid psil-HBV was constructed and transfected into HepG2.2.15 cells. At 48, 72 and 96h after transfection, culture media were collected and cells were harvested for HBV replication assay. HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. Intracellular viral DNA and covalently closed circular DNA (cccDNA) was quantified by real-time PCR. HBV viral mRNA was measured by reverse-transcript PCR.
  
  
  RESULTS
  Our data demonstrated that the three used siRNAs showed marked anti-HBV effects. Combination of siRNAs, compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication, even though the final concentration of siRNA in the therapy was the same. More importantly, we showed that combination therapy significantly suppressed HBV cccDNA amplification.
  
  
  CONCLUSIONS
  Our results revealed that combination of siRNAs mediated a stronger inhibition on viral replication and antigen expression in HepG2.2.15 cells, especially, the amplification of cccDNA.
authors.affiliation: The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China.
authors.forename: Gui-Qiu
authors.initials: GQ
authors.lastname: Li
authors.forename: Dong-Mei
authors.initials: DM
authors.lastname: Yu
authors.forename: Juan
authors.initials: J
authors.lastname: Lu
authors.forename: Shu-Lan
authors.initials: SL
authors.lastname: Chen
authors.forename: Jin-Ying
authors.initials: JY
authors.lastname: Zhao
authors.forename: Yong-Chen
authors.initials: YC
authors.lastname: Wang
mesh_terms: D004279:DNA, Viral / Q000032:analysis; D018390:Gene Targeting; D056945:Hep G2 Cells; D006514:Hepatitis B Surface Antigens / Q000032:analysis; D006513:Hepatitis B e Antigens / Q000032:analysis; D006515:Hepatitis B virus / Q000235:genetics* / Q000502:physiology; D006801:Humans; D019913:Nuclear Localization Signals; D034741:RNA, Small Interfering / Q000235:genetics*; D014779:Virus Replication
chemical_list: D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D019913:Nuclear Localization Signals; D034741:RNA, Small Interfering