title: Combination of small interfering RNAs mediates greater inhibition of human hepatitis B virus replication and antigen expression.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Cancer Institute, Second affiliated hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Chen
authors.forename: Ze-feng
authors.initials: ZF
authors.lastname: Xu
authors.forename: Jing-jia
authors.initials: JJ
authors.lastname: Ye
authors.forename: Hang-ping
authors.initials: HP
authors.lastname: Yao
authors.forename: Shu
authors.initials: S
authors.lastname: Zheng
authors.forename: Jia-yi
authors.initials: JY
authors.lastname: Ding
mesh_terms: D017209:Apoptosis; D002453:Cell Cycle; D045744:Cell Line, Tumor; D004279:DNA, Viral / Q000096:biosynthesis; D005434:Flow Cytometry; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D006514:Hepatitis B Surface Antigens / Q000378:metabolism*; D006513:Hepatitis B e Antigens / Q000378:metabolism*; D006515:Hepatitis B virus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics*
chemical_list: D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D034741:RNA, Small Interfering