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

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.

Efficient Silencing of Endogenous MicroRNAs Using Artificial MicroRNAs in Arabidopsis thaliana

We report here that the expression of endogenous microRNAs （miRNAs） can be efficiently silenced in Arabi- dopsis thaliana （Arabidopsis） using artificial miRNA （amiRNA） technology. We demonstrate that an amiRNA designed to target a mature miRNA directs silencing against all miRNA family members, whereas an amiRNA designed to target the stem-loop region of a miRNA precursor transcript directs silencing against only the individual family member targeted. Furthermore, our results indicate that amiRNAs targeting both the mature miRNA and stem-loop sequence direct RNA silencing through cleavage of the miRNA precursor transcript, which presumably occurs in the nucleus of a plant cell during the initial stages of miRNA biogenesis. This suggests that small RNA （sRNA）-guided RNA cleavage in plants occurs not only in the cytoplasm, but also in the nucleus. Many plant miRNA gene families have been identified via sequencing and bio- informatic analysis, but, to date, only a small tranche of these have been functionally characterized due to a lack of ef- fective forward or reverse genetic tools. Our findings therefore provide a new and powerful reverse-genetic tool for the analysis of miRNA function in plants.

An in vivo Transient Expression System Can Be Applied for Rapid and Effective Selection of Artificial MicroRNA Constructs for Plant Stable Genetic Transformation

The utility of artificial microRNAs（amiRNAs） to induce loss of gene function has been reported for many plant species,but expression efficiency of the different amiRNA constructs in different transgenic plants was less predictable,In this study,expressions of amiRNAs through the gene backbone of Arabidopsis miR168a were examined by both Agrobacterium-mediated transient expression and stable plant genetic transformation.A corresponding trend in expression of amiRNAs by the same amiRNA constructs between the transient and the stable expression systems was observed in the experiments.Plant genetic transformation of the constructs that were highly expressible in amiRNAs in the transient agro-infiltration assays resulted in generation of transgenic lines with high level of amiRNAs.This provides a simple method for rapid and effective selection of amiRNA constructs used for a time-consuming genetic transformation in plants.