Hepatitis B virus preS2 gene-specific locked nucleic acid antisense oligonucleotides significantly inhibit hepatitis B virus replication and expression in HepG2 2.2.15 cells

Abstract

AIM: To investigate the inhibitory effects of hepatitis B virus (HBV) preS2 gene-specific locked nucleic acid (LNA) antisense oligonucleotides on HBV replication and expression in HepG2 2.2.15 cells. METHODS: Three LNA antisense oligonucle-otides of different lengths that are complementary to the translation initiation region of the HBV preS2 gene were designed, synthesized and introduced into HepG2 2.2.15 cells by cationic liposome-mediated transfection. Hepatitis B surface antigen (HbsAg) and HBV DNA levels in cell supernatant were tested by time-resolved immunofluorescence assay (TRFIA) and fluorescent quantitative-polymerase chain reaction (FQ-PCR). The inhibitory effects of different antisense oligonucleotides on HBV DNA replication and expression were compared. The cell toxicity of LNA antisense oligonucleotides was evaluated by methyl thiazolyl tetrazolium (MTT) assay. RESULTS: On day 1 after transfection with LNA antisense oligonucleotides, the expression of HBsAg and the replication of HBV DNA were inhibited. On day 7, the reduced rates of HBsAg and HBV DNA levels were 45.79%, 52.92% and 67.21% as well as 35.15%, 40.69% and 52.16% in the non-modified antisense oligonucleotide group, all-phosphorothioate-modified antisense oligonucleotide group and LNA antisense oligo-nucleotide group, respectively. LNA antisense oligonucleotides showed the strongest inhibitory effects on viral activity and had no impact on cell metabolism. Compared with the control group, the reduced rates of HBsAg and HBV DNA levels achieved in each of the above groups were significantly higher (all P＜0.01). Moreover, the reduced rates of HBsAg and HBV DNA levels in the LNA antisense oligonucleotide group were significantly higher than those in other antisense oligonucleotide groups (all P＜0.05). CONCLUSION: LNA antisense oligonucleotides targeting the preS2 gene can effectively inhibit the replication and expression of HBV in vitro. The preS2 gene can be used as an effective target for gene therapy of HBV infection.