title: Combination of small interfering RNAs mediates greater suppression on hepatitis B virus cccDNA in HepG2.2.15 cells.
abstract: OBJECTIVE
  To observe the inhibition of hepatitis B virus (HBV) replication and expression in HepG2.2.15 cells by combination of small interfering RNAs (siRNAs).
  
  
  METHODS
  Recombinant plasmid psil-HBV was constructed and transfected into HepG2.2.15 cells. At 48 h, 72 h and 96 h 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 (ELISA). Intracellular viral DNA and covalently closed circular DNA (cccDNA) were quantified by real-time polymerase chain reaction (PCR). HBV viral mRNA was reverse transcribed and quantified by reverse-transcript PCR (RT-PCR).
  
  
  RESULTS
  siRNAs showed marked anti-HBV effects. siRNAs could specifically inhibit the expression of HBsAg and the replication of HBV DNA in a dose-dependent manner. Furthermore, combination of siRNAs, compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication. More importantly, combination of siRNAs significantly suppressed HBV cccDNA amplification.
  
  
  CONCLUSIONS
  Combination of siRNAs mediates a stronger inhibition on viral replication and antigen expression in HepG2.2.15 cells, especially on cccDNA amplification.
authors.affiliation: Department of Laboratory Diagnosis, the First Affiliated Hospital of Harbin Medical University, Heilongjiang Province, Harbin, China.
authors.forename: Xiao-Min
authors.initials: XM
authors.lastname: Xin
authors.forename: Gui-Qiu
authors.initials: GQ
authors.lastname: Li
authors.forename: Ying-Yu
authors.initials: YY
authors.lastname: Jin
authors.forename: Min
authors.initials: M
authors.lastname: Zhuang
authors.forename: Di
authors.initials: D
authors.lastname: Li
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000276:immunology / Q000378:metabolism / Q000821:virology*; D045744:Cell Line, Tumor; D004279:DNA, Viral / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D016174:Hepacivirus / Q000235:genetics* / Q000502:physiology; D006514:Hepatitis B Surface Antigens / Q000187:drug effects / Q000378:metabolism; D006513:Hepatitis B e Antigens / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000276:immunology / Q000378:metabolism / Q000821:virology*; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D014162:Transfection; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D012333:RNA, Messenger; D034741:RNA, Small Interfering