Study On Optimized Tandem AmiRNA Inhibitory Effects On Hepatitis B Virus Infection

It is well known that as an infectious disease, hepatitis B is now threatening to human health seriously. Although there are safe and effective vaccines can protect the crowd. The vaccines are invalid to chronic HBV patients. Chronic HBV infection is associated with high risk of liver cirrhosis and primary hepatocellular carcinoma (HCC). In HBV-infected individuals, so long as serum HBV viral levels are undetectable, patient condition, including pathological indices, will improve.Currently, interferons and nucleoside analogs, which act as immunomodulators and viral polymerase inhibitors respectively, are the principal treatment options for chronic HBV infection. However, these therapies have many limitations. Therefore, novel and improved strategies are in urgent demand for the treatment of HBV infection. RNA interference (RNAi) has emerged as a potential new approach against HBV infection, after antisense RNA and ribozyme skills.RNA interference (RNAi) provides a promising therapeutic approach to human diseases. Some researches had been done about RNAi inhibiting HBV. But each team obtained different inhibitory effect. Therefore a lot of work should be done to search the optimal targets and the designing method. Inhibition of HBV by RNAi method must achieve longer-lasting suppression in order to be clinically relevant. Even a single point mutation (mismatch) in the virus is sufficient to confer escape from siRNA inhibition. In contrast, microRNAs (miRNAs) do not require exact sequence complementation, and thus miRNA-based therapeutic gene silencing is more desirable for combating highly mutable viruses. Some researchers had tested the feasibility of combination treatment of Chemical synthetic siRNAs targeting various regions and the antiviral efficacy of HepG2.2.15 cell culture system. But it’s dose dependent, and we must often inject to body with siRNA. The plasmid vector can mediate long-term interference effect, but it may cause certain effect to cells. Instead of introducing more vectors, tandem sequences technique just need one vector. It is reported that multiple shRNAs expressed by an inducible pol II promoter could knockdown the expression of multiple target genes. Relative to siRNA, miRNA method caused no detectable toxicity that result from IFN response induction or disruption of the endogenous miR pathway. Researchers found that miRNA-based therapies are better suited for therapeutic silencing in vivo. Some people inserted two anti-HBV miRNA sequences in the miRNA polycistron, but the interference effect didn’t surpass the single one. Maybe it is because that these are not the better sites. Furthermore the interference effect at HBV DNA and proteins levels especially cccDNA were not detected. HBV is a DNA virus which replicates by reverse transcription of a pre-genomic RNA intermediate. cccDNA is the source of HBV, which make it more important.Objective:Based on the experience of previous researches, HBV genome can vary easily and escape from siRNA inhibition. In this study, we try to seek the efficient sites in HBV genomic conservative regions. We constructed expression plasmid vectors of four distinct amiRNA. We also believe that using a pool of miRNAs to simultaneously target multiple sites in the viral genome could achieve better gene silencing as well as more effectively prevent the emergence of resistant virus mutants. To this end, we devised our expression amiRNA in tandem, mimicking naturally occurring tandem miRNAs. Through the transient transfection assays and stable screening, a comprehensive study about amiRNA expression vector in HBV RNA、DNA、protein and the cccDNA aspects was made.Methods:1. For the design of artificial microRNA (amiRNA) sequences,15 HBV genomes were aligned with the online basic local alignment search tool to identify the conserved region, which was then selected as the target sequences of miRNAs.2. We used miRNA expression vector that expresses miRNA in a murine miRNA skeleton so as to avoid the stimulation of interferon production such as in the cases of SiRNA models. Four miRNAs were designed; they were named as amiHBV-1, amiHBV-2, amiHBV-3, and amiHBV-4. We checked by BLAST to prevent non-specific off-target inhibition.3. The four constructed distinct singular miRNAs were transfected into HepG2.2.15 cells by liposome. After transfection, expression of HBV mRNA was assessed using Q-PCR analysis. RNA was prepared 72 h post-transfection and subjected to reverse transcription and quantitative PCR analysis. When compared to negative controls, two sequences with high efficiencies were selected to build the tandem one (amiHBV 3-4)4. Next we evaluated the inhibitory effect of miRNA vectors on HBV DNA and protein expression of singular and tandem ones. Since ami-HBV1 and ami-HBV2 demonstrated only moderate effect on HBV mRNA expression, they were dropped from further analysis. Using real time quantitative PCR and CMIA method, respectively. Due to only minimal to modest inhibition effects, amiHBV 1 and amiHBV 2 were dropped for further analysis. The concentrations of HBsAg and HBeAg in the culture supernatant were measured at 24h,48h,72h and 96h after transfection using CMIA method, ideally suitable for measuring secretory proteins with high sensitivity.5. We screened cells which stably expressed miRNA. cccDNA were measured by real time quantitative PCR to observe the long-term inhibition efficiency.Results:1. Four miRNAs were designed. The oligos were cloned into pcDNATM 6.2-GW/±EmGFP-miR(Invitrogen) to construct four miRNA expression vectors(pcDNA6.2-GW/ EmGFP-ami-HBV 1,2,3,4 were abbreviated as ami-HBV-1, ami-HBV-2, ami-HBV-3, and amiHBV-4). Then, a tandem sequence miRNA expression vector was constructed. They were all verified by DNA sequencing.2. In transient transfection assays, the inhibition at HBV RNA level was 29.3%, 14.9%,61.2%,75.6% for the four monomers, and 87.2% for the tandem dimer.3. Since ami-HBV1 and ami-HBV2 demonstrated only moderate effect on HBV mRNA expression (29.3% or 14.9% inhibition, respectively), they were dropped from further analysis. We examined the inhibitory effect of miRNA vectors on HBV DNA level as a representation of HBV replication. When we measured HBV DNA level in the supernatants of HepG2.2.15 culture, as much as 94.3% reduction of HBV DNA level was detected in tandem amiRNA-HBV3-HBV4 transfected cells, but only 60.5% and 68.0% reduction were detected in amiRNA-HBV3 and amiRNA-HBV4 transfected cells, respectively. Furthermore, we measured intracellular HBV DNA levels in amiRNA transfected cells, and observed 71.6%,80.2% and 79.7% reduction of HBV DNA levels in amiRNA-HBV3, amiRNA-HBV4 and amiRNA-HBV3-HBV4 transfected cells at 96 h post-transfection, respectively.4. Next we evaluated the inhibitory effect of miRNA vectors on HBV protein expression. For HBsAg, inhibition can be detected at 24 h post transfection, reached plateau at 72 h post transfection. HBsAg inhibition ranking was amiHBV 3-4(67.4%)> amiHBV 4(64.6%)> amiHBV 3(52.3%). For HBeAg, overall inhibition effects lack in comparison to those of HBsAg. After Stable transfection, two groups (amiHBV 3-4 and amiHBV 4) showed significant inhibitory effect on HBsAg (97.18%) and HBeAg secretion (97.00%)5. cccDNA were measured to observe the long-term inhibition efficiency. HBV cccDNA was inhibited by 21.9% and 58.4% respectively after instantaneous transfection of amiHBV 4 and amiHBV3-4 plasmids. Later after stable transfection, cccDNA remarkably decreased in amiHBV 4 group by 93.78% compared to the negative control plasmid group; amiHBV 3-4 group by 99.65%.Conclusion:In the present study, we constructed expression plasmid vectors of four distinct monomeric amiRNA and one heterodimeric tandem amiRNA. The amiRNA can specifically target the S antigen and X gene of conservative regions of HBV. Because of the limitation of polⅢ, we utilized the vector which Based on natural miR-155 structure skeleton containing polⅡpromoter and express endogenous miRNA. After the construction of the recombinant expression vectors, they were confirmed by enzyme digestion, electrophoresis and gene sequencing.The amiRNA plasmids of singular and tandem sequences against HBV were transfected into HepG2.2.15 transiently. Then we screened cells which stably expressed miRNA. We analyzed that miRNA-mediated HBV suppression at DNA (including cccDNA) and protein (viral antigen) levels are significant. Targeting S area of HBV gene maybe is one of the effective methods of RNA interference. The inhibition effects after stable expression of miRNA were superior to instantaneous transfections. Our results proved that multiple miRNAs in tandem aimed at multiple targeting sites is effective than single ones.In this regard, amiRNA-based therapeutic gene silencing is more advantageous in combating highly mutable viruses. We paved the way for this plasmid expressing carrier to further study in vivo.