Combination therapy of siRNAs mediates greater suppression on hepatitis B virus cccDNA in HepG2.2.15 cell.
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 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. Intracellular viral DNA and covalently closed circular DNA (cccDNA) was quantified by real-time PCR. HBV viral mRNA was reverse transcribed and quantified by reverse-transcript PCR.


RESULTS
Our data demonstrated that three used siRNAs showed marked anti-HBV effects. The expression of HBsAg and the replication of HBV DNA could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, 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.
Xin
Xiao-min
XM
Department of Microbiology, Harbin Medical University, Harbin 150081,Heilongjiang Province, China.
Li
Gui-qiu
GQ
Guan
Xiu-ru
XR
Li
Di
D
Xu
Wei-zhen
WZ
Jin
Ying-yu
YY
Gu
Hong-xi
HX
D006528:Carcinoma, Hepatocellular / Q000276:immunology / Q000821:virology*; D045744:Cell Line, Tumor; D004279:DNA, Viral / Q000187:drug effects* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006514:Hepatitis B Surface Antigens / Q000378:metabolism; D006513:Hepatitis B e Antigens / Q000378:metabolism; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D008113:Liver Neoplasms / Q000276:immunology / Q000821:virology*; D010957:Plasmids; D034741:RNA, Small Interfering / Q000494:pharmacology*; D014162:Transfection; D014779:Virus Replication / Q000187:drug effects* / Q000235:genetics
D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D034741:RNA, Small Interfering