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

Abstract

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 artificial microRNA targeting the HBV S coding region. Vector-based artificial microRNA could be a promising therapeutic approach for chronic HBV infection. INTRODUCTIONRNA interference (RNAi) has become a new promising approach to develop effective antiviral drugs in recent years, including hepatitis C vir us (HCV), human immunodeficiency virus (HIV), poliovirus and hepatitis B vir us (HBV) [1][2][3] . For HBV, RNAi was shown to have impressive inhibitory effects against viral gene transcription and expression [4][5][6][7] . One recent study further demonstrated the viral clearance from the liver of transgenic mice by RNAi targeted HBV [8] , which sheds light on the use of RNAi in HBV gene therapy.HBV replication and gene expression can be strongly inhibited with virus specific siRNA treatment. However, the high sequence specificity of siRNAs, combined with prolonged treatment, promoted the emergence of siRNA-resistant virus variants. Selection of RNAi 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 < escape mutants has been reported in vitro for HIV, HCV and HBV [9][10][11] . These findings indicate that the antiviral properties of specific siRNAs targeted virus are not as effective as expected.RNAi can be triggered by small RNA molecules such as siRNA and microRNAs (miRNAs). MiRNAs endogenously expressed small ssRNA sequences of about 22 nucleotides in length, which naturally direct gene silencing through components shared with the RNAi pathway [12] . The mature miRNAs regulate gene expression by mRNA cleavage or translational repression [13,14] . Compared with siRNA, miRNA still can play a translational repression role when miRNAs partially complement with the target gene. Because of the flexibility of miRNA in binding with partially complementary mRNA targets, miRNA can serve as an anti-virus drug or vaccine to achieve a breakthrough in the treatment of virus mutation. Moreover, since miRNAs are single-stranded molecules insensitive to interferon systems, the utilization of this Pol-Ⅱ-mediated miRNA generation can be safe both in vitro and in vivo without the cytotoxic effects of dsRNAs and siRNAs [15] . These findings indicate that miRNA mediated RNAi can be used...

MicroRNAs and hepatitis viruses

“…In 2009, it was also found that miRNA-199a played an important role in inhibiting HCV genome replication and may also serve as a novel antiviral therapy [39]. In the same way, vectorbased artificial miRNA that was transfected into HeG2.2.15 cells could inhibit HBV replication, and could be a promising therapeutic approach for HBV infection [40]. Not just the mature miRNA, but pol IIexpressed pri-miR mimics may be useful in the treatment of HBV infection [41].…”

“…Artificial miRNA-mediated RNA interference (RNAi), based on the murine miR-155, could inhibit HBV protein expression and HBV DNA replication in HepG2.2.15 cells in vitro [15]. In mammals, miR-122 is abundant in liver to help maintain liver tissue identity [16].…”

RNA interference and its therapeutic potential

“…Immunohistochemical detection of HBV core antigen revealed 99% reduction in stained hepatocyres upon RNAi treatment [50]. It was also noticed that HVB replication and expression has been inhibited by artificial microRNA targeting the HBV S coding region in HepG2.2.15 cells [27]. This vector-based artificial microRNA could be a promising therapeutic approach for curing chronic HBV infection.…”

The Therapeutic Potential of MicroRNAs: Disease Modulators and Drug Targets

“…The authors proposed that HBV had evolved to use viral miRNAs as a means to regulate its own gene expression to its benefit (40). This hypothesis was confirmed in vitro when vector-based artificial miRNA (amiRNA) successfully inhibited HBV replication and expression (41). Ely et al confirmed the in vivo viability of this potential therapeutic approach to HBV by employing RNA polymerase II promoter cassettes that transcribes anti-HBV primary miRNA shuttles, specifically pri-mi-122 and pri-mi-31, with a resulting decreases in HBV expression (42).…”