Inhibition of hepatitis B virus gene expression and replication by artificial microRNA

• Published in: World journal of gastroenterology : WJG 2008-08, Vol.14 (29), p.4684-4689
• Main Authors: Gao, Yu-Feng, Yu, Li, Wei, Wei, Li, Jia-Bin, Luo, Qing-Li, Shen, Ji-Long
• Format: Article
• Language: English
• Subjects: Carcinoma, Hepatocellular - metabolism; Carcinoma, Hepatocellular - pathology; Carcinoma, Hepatocellular - virology; Cell Line, Tumor; DNA Replication - drug effects; DNA, Viral - genetics; Gene Expression Regulation, Viral - drug effects; Hepatitis B e Antigens - metabolism; Hepatitis B Surface Antigens - metabolism; Hepatitis B virus - genetics; Hepatitis B virus - immunology; Hepatitis B virus - physiology; Humans; Liver Neoplasms - metabolism; Liver Neoplasms - pathology; Liver Neoplasms - virology; MicroRNAs - pharmacology; Plasmids; Rapid Communication; RNA, Messenger - metabolism; RNA干扰; Transfection; Virus Replication - drug effects; 乙型病毒肝炎; 人造基因; 病毒感染
• ISSN: 1007-9327; 2219-2840
• DOI: 10.3748/wjg.14.4684

AIM： 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 ？ uorescence 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. CONCLUSION： In HepG2.2.15 cells, HBV replication and expression could be inhibited by artif icial microRNA targeting the HBV S coding region. Vector-based artificial microRNA could be a promising therapeutic approach for chronic HBV infection.