| Chronic hepatitis B virus (HBV) infection remains an important world-wide public health problem. The number of persons with chronic HBV infection has exceeded 350 millions all over the world. It is one of the leading causes of liver cirrhosis and hepatocellular carcinoma(HCC). China is the highly affected area in which 10 percent of populations are infected with HBV. Current treatment strategies of HBV have met with only partial success. Therefore, it is necessary to develop more effective antiviral therapies that can clear HBV infection with fewer side effects.RNA interference (RNAi) is a new developmental gene silencing technology. It is a natural process by which double-stranded RNA directs sequence-specific post-transcriptional silencing of homologous genes. RNAi can be triggered by small RNA molecules such as siRNA and miRNA. However, siRNA mediated RNAi silences gene expression in a sequence-specific manner through the actions of small pieces of double-stranded RNAs. Many viruses escape RNAi-mediated suppression by counteracting the RNAi machinery through mutation of the targeted region, by encoding viral suppressors, or both. MicroRNAs (miRNAs) are endogenously expressed small ssRNA sequences of ~22 nucleotide in length, which naturally direct gene silencing through components shared with the RNAi pathway. The mature miRNAs can regulate gene expression by mRNA cleavage or translational repression. Compared with siRNA, miRNA still can play a translational repression role when miRNA complement partly 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 treatments of viral mutations. The study of amiRNA functions will be helpful for improving HBV therapy.Given these circumstances, an alternative approach to antiviral siRNA therapy is to target one or more cellular genes making products that help to support virus replication. HBV infection and replication in vivo need the participation of host cellular genes. It is possible that RNAi targeting host cellular genes can decrease the replication of HBV. The advantage of RNAi targeting host genes is not depended on virus gene, so it can escape virus mutation in RNAi.Recently a large number of studies show that amiRNA, which targeted specific gene, can be introduced into cells through microRNA expression framework, and can inhibit the expression of target genes. Therefore, this experimental study constructed amiRNA expression plasmids which targeted different HBV sites,ASGPR1 gene and La protein genes to transiently and stable transfected HepG2.2.15 cells. We observed whether amiRNA-mediated RNA interference could block replication of HBV and HBV-related host gene expression by measuring the inhibition effect of the target gene, HBV DNA, and protein decrease level, so that we could understand whether amiRNA mediated RNAi targeting HBV and related host gene can be used in HBV gene therapy. Objective: The amiRNA vectors which targeted HBV different sites,ASGPR1 gene and La protein genes were constructed by untilizing microRNA expression framework, then were transfected into HepG2.2.15 cells. The aims of this study were to observe whether amiRNA-mediated RNA interference could decrease the replication level of HBV.Method: Thirteen amiRNA expression vectors were constructed based on the this study, the inserted DNA sequences of recombinant vectors mentioned above were all correct by DNA sequencing. The plasmids were transient and stably transfected by lipofectemine 2000 reagment. semi-quantitative RT-PCR was utilized to observe suppression level of the target gene mRNA; Western blot was utilized to observe suppression level of the target gene proteins. The MEIA was carried out to detect quantitatively HBsAg and HBeAg, the fluorescent quantitation PCR was utilized to observe the changes of HBV DNA copies.Results:1. The inhibition of HBsAg and HBeAg by transient transfected amiRNA-HBV.The seven amiRNA expression plasmid were transfected into HepG2.2.15 cells. The transfection efficiency were 50%~60% by FCM analysis. Under such circumstances, the assay results showed: the three of seven plasmid had obvious effect from 48h post-transfection, the inhibition of the HBsAg and HBeAg at 72h reached a peak, inhibitory effect gradually weakened at 96h post-transfection. The strongest inhibitor among the seven vectors, compared with mock group, the secretion of HBsAg in the supernatant was inhibited by 30.5%,49.8% and 46.3% respectively at 48h, 72h and 96h after amiRNA-HBV-S608 plasmids transfection, P<0.01, the secretion of HBeAg in the supernatant was inhibited by 25.5%,39.9% and 37.4% respectively at 48h, 72h and 96h after amiRNA-HBV-P2227 plasmids transfection, P<0.01. However, the change of negative control group was not obvious, P>0.05. 2. The inhibition of HBV DNA level by transient transfected amiRNA-HBVReal-time fluorescence quantitative PCR was performed to detrmine HBV DNA level at different time points. Similarly with the decrease degree of HBsAg and HBeAg, seven amiRNA-HBV plasmids resulted in different inhibition degree. Quantitative assay revealed that the greatest reduction was seen in the amiRNA-HBV-P2227 transfection group. The copies of HBV DNA in cells treated with amiRNA-HBV-P2227 was reduced by35.7%,48.2% and 39.5% at 48h, 72h and 96h respectively compared to mock controls, P<0.01.3. The inhibition of HBsAg and HBeAg level by stable transfected amiRNA-HBVHBsAg and HBeAg in stable transfected cells culture medium were assayed. The HBsAg levels of all the three cell lines integrated with the pcDNA6.2-HBV-amiRNA vector were significantly reduced compared with HepG2.2.15 cells, and the greatest reduction on amiRNA-S608 is 81.3%, P<0.01. HBeAg was also reduced in stable transfected cells, and the greatest reduction on amiRNA-S608 was 74.9%, P<0.01. No significant reduction was measured on the cells stably transfected with negative control compared with 2.2.15 cells, P>0.05.4. The inhibition of HBV DNA level by stable transfected amiRNA-HBVFor stable transfected cell clones, compared with negative control vector, amiRNA-mediated RNAi resulted in a higher reduced level of HBV DNA in three plasmids transfected cells than transient transfection. Among them, amiR-HBV-P2227 and amiRNA-HBV-X402 was much more efficient with 76.3% and 67.9% inhibitory rate, P<0.01.5. The inhibition of HBV replication and expression level by amiRNA targeted La gene.The results showed that: compared with mock control, HepG2.2.15 cell proliferation and cell growth rate had no significant difference. In La mRNA and protein level, the results showed that three amiRNA can significantly reduce the La mRNA and protein expression, the greatest reduction on amiRNA-La-1130 transfected group, the expression of La mRNA and protein levels were down-regulation by 58.6% and 50.6% after 72 h, P<0.01. In the virus level, the one of three amiRNA-La plasmids could obviously inhibit the secretion of HBsAg and HBeAg, the greatest reduction on amiRNA-La-1130 transfected group, the expression of HBsAg and HBeAg were down-regulation by 23.3% and 25.8% at 72 h, P<0.01, HBV DNA were down-regulation by 22.3% after 72 h, P<0.01.6. The inhibition of HBV replication and expression level by amiRNA targeted ASGPR1 gene.The results showed that: compared with mock control, HepG2.2.15 cell proliferation and cell growth rate had no significant difference. In La mRNA and protein level, the results showed that three amiRNA can significantly reduce the La mRNA and protein expression, the greatest reduction on amiRNA-ASGPR1-610 transfected group, the expression of La mRNA and protein levels were down-regulation by 57.3% and 49.8% at 72 h, P<0.01. In the virus level, the two of three amiRNA-ASGPR1 plasmids could obviously inhibit the secretion of HBsAg and HBeAg, the greatest reduction on amiRNA-ASGPR1-610 transfected group, the expression levels of HBsAg and HBeAg were down-regulation by 31.3% and 33.6% after 72 h, P<0.01, HBV DNA level was down-regulation by 29.7% at 72 h, P<0.01.Conclusion:1. Artificial microRNA-mediated RNA interference can be high effectively block HBV expression in specific manners.2. Artificial microRNA-mediated RNA interference can block La and ASGPR1 host target gene expression. 3. Host La and ASGPR1 gene expression level is closely related with HBV replication and expression, furtherly confirmed that the two gene play a key role in the HBV infection and replication. The relevant host genes of HBV infection and replication could be an optional targets for HBV gene therapy.4. Improving the efficiency of transfection, artificial microRNA could be act as a new means of HBV gene therapy...
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