Inhibition of hepatitis B virus gene expression and replication by artificial microRNA.
OBJECTIVE
To investigate the inhibitory effects of hepatitis B virus (HBV) replication and expression by transfecting artificial microRNA (amiRNA) into HepG2.2.15 cells.


METHODS
Three amiRNA-HBV plasmids were constructed and transfected into HepG2.2.15 cells. HBV antigen secretion was detected in the cells with transient and stable transfection by time-resolved fluoroimmunoassays (TRFIA). HBV DNA replication was examined by fluorescence quantitative PCR, and the level of HBV S mRNA was measured by semi-quantitative RT-PCR.


RESULTS
The efficiency of transient transfection of the vectors into 2.2.15 cells was 55%-60%. All the vectors had significant inhibition effects on HBsAg and HBeAg at 72 h and 96 h after transfection (P < 0.01 for all). The secretion of HBsAg and HBeAg into the supernatant was inhibited by 49.8% +/- 4.7% and 39.9% +/- 6.7%, respectively, at 72 h in amiRNA-HBV-S608 plasmid transfection group. The copy of HBV DNA within culture supernatant was also significantly decreased at 72 h and 96 h after transfection (P < 0.01 for all). In the cells with stable transfection, the secretion of HBsAg and HBeAg into the supernatant was significantly inhibited in all three transfection groups (P < 0.01 for all, vs negative control). The copies of HBV DNA were inhibited by 33.4% +/- 3.0%, 60.8% +/- 2.3% and 70.1% +/- 3.3%, respectively.


CONCLUSIONS
In HepG2.2.15 cells, HBV replication and expression could be inhibited by artificial microRNA targeting the HBV S coding region. Vector-based artificial microRNA could be a promising therapeutic approach for chronic HBV infection.
Gao
Yu-Feng
YF
Institute of Clinical Pharmacology, Anhui Medical University, Hefei, Anhui Province, China.
Yu
Li
L
Wei
Wei
W
Li
Jia-Bin
JB
Luo
Qing-Li
QL
Shen
Ji-Long
JL
D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology / Q000821:virology; D045744:Cell Line, Tumor; D004261:DNA Replication / Q000187:drug effects; D004279:DNA, Viral / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000187:drug effects*; D006514:Hepatitis B Surface Antigens / Q000378:metabolism; D006513:Hepatitis B e Antigens / Q000378:metabolism; D006515:Hepatitis B virus / Q000235:genetics* / Q000276:immunology / Q000502:physiology*; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology / Q000821:virology; D035683:MicroRNAs / Q000494:pharmacology*; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection; D014779:Virus Replication / Q000187:drug effects*
D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D035683:MicroRNAs; D012333:RNA, Messenger