[Inhibition of HBV DNA replication and expression in 2.2.15 hepatoma cells infected with AFP-mediated HBX antisense RNA].
OBJECTIVE
To study the specific expression of the antisense RNA against hepatitis B virus X (HBX) gene in hepatoblastoma cell line and its anti -HBV activity.


METHODS
HBX gene (nt.1370-1827) was amplified by PCR, then cloned into EB virus vector pEBAF which contained human alpha-fetoprotein promoter and enhancer. After transfected into 2.2.15 hepatoma cells and ECV304 human endothelial cells by lipofectin, northern blot, ELISA and real-time qualitative PCR were carried out to assay the expression of HBX mRNA, HBV antigens and HBV DNA level, respectively.


RESULTS
The HBX antisense RNA expression vector pEBAF-as-HBX which could be expressed specifically in 2.2.15 hepatoblastoma cells was successfully constructed. Both HBV DNA level and the expressions of hepatitis B virus surface antigen (HBsAg) and e antigen (HBeAg) in 2.2.15 hepatoblastoma cells were inhibited by pEBAF-as-HBX. Compared with those in sense control (pEBAF-s-HBX), the inhibitory rates of HBsAg, HBeAg, and HBV DNA were 37.9%, 36.8%, and 25%, respectively.


CONCLUSIONS
The pEBAF-as-HBX expression vector may lead to targeted-expression of HBX antisense RNA in hepatoma cells and shows great inhibition effect on HBV.
Ma
Chun-hong
CH
Institute of Immunology, Medical School of Shandong University, Jinan 250012, China.
Sun
Wen-sheng
WS
Liu
Su-xia
SX
Wang
Xiao-yan
XY
Zhang
Li-ning
LN
Cao
Ying-lin
YL
Han
Li-hui
LH
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology / Q000821:virology*; D045744:Cell Line, Tumor; D004261:DNA Replication; D004742:Enhancer Elements, Genetic / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D015316:Genetic Therapy / Q000379:methods; D006515:Hepatitis B virus / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology / Q000821:virology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D016372:RNA, Antisense / Q000494:pharmacology*; D015534:Trans-Activators / Q000096:biosynthesis* / Q000235:genetics; D015533:Transcriptional Activation; D014162:Transfection; D054334:Viral Regulatory and Accessory Proteins; D000509:alpha-Fetoproteins / Q000235:genetics*
D016372:RNA, Antisense; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; D000509:alpha-Fetoproteins; C057628:hepatitis B virus X protein