Inhibition Of HBV Replication And Expression By RNAi

Hepatitis B virus (HBV) is one of the most important infectious pathogens causing viral hepatitis. With more then 350 million people infected, Hepatitis B is widely prevalent and severely harmful to human health. Chronic HBV infection can lead to the development of liver cirrhosis and hepatocellular carcinoma, which substantially threat health of people in China. Although hepatitis B vaccines have been used for many years worldwide, HBV infection is still a big challenge to many countries. Until now, interferon-α and nucleoside analogs, such as lamivudine, are of the few drugs capable of inhibiting HBV replication. However, incomplete responses, relapses and escape mutants all limit their usefulness. So, the need for new effective anti-HBV therapies is pressing. Gene therapy for HBV infection, such as antioligonucleotide, ribozyme and deoxyribozyme, has made rapid progress in recent years, still with some problems involved. The HBV genome is a partially double-stranded DNA molecule which replicates by reverse transcription of a pre-genomic RNA intermediate in a manner similar to that of the retroviruses. The3.5-kb mRNA not only represents the template for reverse transcription but also serves for translation of the virus proteins. HBV genome overlaps obviously so that the synthesis of HBV proteins could be effectively inhibited by cutting its mRNA and pregenomic RNA on proper site.Recently, the appearance of RNA interference (RNAi) has opened the door for a number of novel anti-HBV therapies. RNAi, also called gene silencing or post-transcriptional gene silencing ( PTGS ) , is a process during which double-stranded RNA (dsRNA) induces the sequence-specific degradation of homologous messenger RNA (mRNA). As a result, the expression of the target gene is suppressed and becomes undetectable. Because of its high specificity and efficacy, RNAi has been widely used in the field of gene function investigation in plants, fungi, helminthes, and invertebrates and, more recently, mammalian. Lots of investigations show that RNAi-based anti-HBV replication strategy may represent a potentially efficacious approach to the clinical management of HBV infection.RNAi is initiated by the dsRNA-specific endonuclease Dicer, which promotes processive cleavage of long dsRNA into 21—23 nt (nucleotide, nt) short interfering RNA (siRNA) . The siRNA can lead to specific and effective target mRNA degradation but short enough to avoid unspecific antiviral interferon response in mammalian. To date, with the advances in the techniques of siRNA preparation, RNAi has been used effectively to inhibit the replication and expression of several different pathogenic human viruses in culture, including poliovirus, human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus, etc. Recently, HBV is reported to be susceptible to siRNA not only at the level of post-transcription, but also at the level of replication.HepG2.2.15 cell line, a derivative of the human HepG2 hepatoma cell line,contains integrated HBV ayw DNA. This cell line can stably produce HBV mRNAs, antigens and viral particles and serve as a satisfactory in vitro model for investigation of HBV infection. In the present study, HepG2.2.15 cell line was utilized to observe the effect of RNAi on HBV replication. We developed siRNAs that specifically target the S antigen of HBV and explored the inhibitory effect of siRNAs on HBV replication and expression. The aim of this study was to provide some evidences for anti-HBV therapy by siRNA.Based on the sequence of HBV in GenBank, three siRNAs targeting the S antigen of HBV were synthesized and cloned into pSilencer ZY1, pSilencer ZY2, pSilencer ZY3. At the same time, unrelated siRNA was designed as control. After the construction of the recombinant expression vectors was confirmed by enzyme digestion, electrophoresis and gene sequencing, transfection into HepG 2.2.15 cells was carried out by Metafectene. The expression of HBsAg and HBeAg in the supernatant of HepG2.2.15 cells was assayed by microparticle enzyme immunoassay (MEIA), HBV S-mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR) and the HBV DNA in the supernatant of HepG 2.2.15 cells was examined by Fluorescence Quantitative PCR(FC-PCR). The inhibitory effect of HBV gene expression and replication by the three siRNAs was observed with different concentration of siRNAs and at the different time point post-transfection. The results are as following:1. Regularity of HBsAg and HBeAg secretion in the supernatant of HepG 2.2.15 cells: One day after HepG 2.2.15 cells plating, HBsAg and HBeAg could be detected in cell culture media. The amount of HBsAg and HBeAg secretion increased with plating time prolonging. The peak was at 6~9th days after plating. From then on, the antigen secretion no longer changed.2. Inhibition of HBsAg and HBeAg by three siRNAs targeting the S antigen of HBV: The three recombinant expression vectors were transfected into HepG2 2.2.15 cells. The expression of HBsAg and HBeAg in the supernatant was inhibited at the different time point post-transfection. The peak of inhibitory effect was at 72h. The inhibitory rates of three siRNAs on HBsAg are 81%, 29% and 78% at 24lK48h>72h, and on HBeAg are 35%, 3% and 49% at 241u 481k 72h respectively. Among them, the inhibitory effects of ZYl siRNA and ZY3 siRNA were higher than that of ZY2 siRNA. ZYl siRNA showed the greatest effect to decreases the expression of HBsAg and HBeAg on 6th, with inhibitory rates of 94% and 59% respectively. While on 18th day, ZYl siRNA inhibitory rates of HBsAg and HBeAg reduced to 48% and 27% respectively. At the same time, siRNAs inhibition of HBsAg by indirect immumofluorescence assay (IFA) was observed. Compared with control group, fluorescence-marked HBsAg in ZYlsiRNA-transfected HepG 2.2.15 cells significantly decreased on 6th day post-transfection.3. Inhibition of HBV-DNA by three siRNAs targeting the S antigen of HBV: The three recombinant expression vectors were transfected into HepG 2.2.15 cells and inhibited the replication of HBV-DNA in the supernatant at the different time point post-transfection. The peak of inhibitory effect was at 72h. The inhibitory rates of HBV-DNA are 62%, 31% and 63% at 24h, 481k 72h respectively. Among them, the inhibitory effects of ZYl siRNA and ZY3 siRNA were higher than that of ZY2 siRNA, which indicates not all the siRNAs could suppress HBV expression and replication effectively.4. Inhibition of HBV-S-mRNA by three siRNAs targeting the S antigen of HBV: RT-PCR and Northern blot were carried out to detect HBV S-mRNA 72h post-transfection. Similar to the inhibitory effects of three siRNAs on HBV proteins...