ABSTRACT: Persistent and recurrent infection of hepatitis B virus (HBV) represents one of the most common and severe viral infections
of humans, and has caused a formidable health problem in the affected countries. Currently used antiviral drugs have a very
limited success on controlling HBV replication and infection. RNA interference (RNAi), a process by which double-stranded
RNA (dsRNA) directs sequence-specific degradation of target mRNA in mammalian and plant cells, has recently been used to knockdown
gene expression in various species. In this study, we sought to determine whether RNAi-mediated silencing of HBV viral gene
expression could lead to the effective inhibition of HBV replication. We first developed RNAi vectors that expressed small
interfering RNA (siRNA) and targeted the HBV core or surface gene sequence. Our results demonstrated that these specific siRNAs
efficiently reduced the levels of corresponding viral RNAs and proteins, and thus suppressed viral replication. Treatment
with siRNA gave the greatest reduction in the levels of HBsAg (92%) and in HBeAg (85%) respectively in the cultured cell medium.
Our findings further demonstrated that the RNAi-mediated antiviral effect was sequence-specific and dose-dependent. Therefore,
our findings strongly suggest that RNAi-mediated silencing of HBV viral genes could effectively inhibit the replication of
HBV, hence RNAi-based strategy should be further explored as a more efficacious antiviral therapy of HBV infection.
Chinese Science Bulletin 04/2012; 49(14):1470-1475. · 1.32 Impact Factor
ABSTRACT: In order to provide more efficient transduction of plasmid siRNA into target cells, develop more susceptible transduction into cancer cell types, and more easily explore application in animal experiments, we examined development of an adenoviral vector-mediated siRNA expression system and inhibition of survivin gene expression to induce the growth and apoptosis of hepatocarcinoma cells. A system of adenoviral vector-mediated siRNA expression was constructed for the survivin gene. Survivin gene expression in HepG2 cells infected with recombinant adenovirus was detected by Western blot and RT-PCR, and apoptotic cells were investigated by FAC. Western blot analysis showed that the infection of adenovirus-mediated siRNA against survivin efficiently inhibited the expression of survivin in hepatocarcinoma cells with an inhibitory rate of 66.32%. Semi-quantitative RT-PCR showed that survivin gene mRNA transcription was reduced by nearly 72.34% with a peak at 72 h. The number of apoptotic cells increased. In conclusion, results demonstrated that this adenovirus-mediated siRNA system could serve as a useful tool for both basic research on the analysis of gene function and cancer therapy applications.
Bioscience trends 04/2008; 2(2):88-93. · 0.97 Impact Factor
ABSTRACT: To investigate the anti-HBV effect of fusion protein thymosin alpha1-interferon alpha (TA1-IFN) in vitro and to compare its effect with a combination of interferon alpha and thymosin alpha1.
After 2.2.15 cells were seeded for 24 hours, drugs of five serial concentrations (8000, 4000, 2000, 1000, 500 U/ml) were added to the wells, then the medium was changed every three days. After 2.2.15 cells were treated with drugs for 6 days, the medium was collected. The inhibitory rates on HBsAg and HBeAg were determined using Abbot kit, and the cytotoxicity of different drugs by means of MTT colorimetric assays was also observed.
The inhibitory rate of fusion protein on HBsAg, HBeAg was dose-dependent and reached the maximum at 8000 U/ml concentration. In the meantime, the inhibitory rates of fusion protein on HBsAg and HBeAg were 72.2% +/- 0.8% and 60.4% +/- 1.1% respectively, and the cell survival rate was 85.2% +/- 2.0%; In the corresponding concentration, the inhibitory rates of combination thymosin alpha 1 and interferon alpha on HBsAg and HBeAg were 40.0% +/- 0.7%, 34.5% +/- 3.2% respectively. The results showed significant statistical differences between them; cell survival rate 70.0% +/- 1.9%, and the difference of the results was also significant. Cytotoxicity of fusion protein was weaker than a combination of thymosin alpha 1 and interferon alpha.
Fusion protein TA1-IFN exerted stronger anti-HBV effects in vitro. Its anti-HBV effects in vitro were stronger than the combination of thymosin alpha and interferon alpha, and its cytotoxicity was weaker than the combination of thymosin alpha and interferon alpha. Our studies provided important evidence for clinical research on TA1-IFN, and also brought new hope for hepatitis B therapy.
Zhonghua gan zang bing za zhi = Zhonghua ganzangbing zazhi = Chinese journal of hepatology 05/2005; 13(4):252-4.
ABSTRACT: To construct the plasmid containing short hairpin RNA (shRNA) of survivin in order to suppress the expression of survivin gene in HepG2 and SMMC-7721.
Two 20 to 21 bp reverse repeated motifs of survivin target sequence with 4 bp or 8 bp spacer were synthesized respectively and inserted into plasmid pTZU6+1 to generate the plasmid pshRNA-survivin1 and pshRNA-survivin2; plasmid pEGFP-C1-survivin and pshRNA-survivin1 or pshRNA-survivin2 plasmid were cotransfected into liver cancer cell HepG2 and SMMC-7721 to detect effect of GFP expression respectively and analyze the inhibition of survivin gene.
The recombinant plasmid pshRNA-survivin1 and pshRNA-survivin2 were successfully constructed. The recombinant plasmids suppress the survivin expression by 80% in HepG2 and SMMC-7721.
The result showed that the short hairpin RNA of survivin can efficiently suppress it's expression in HepG2 and SMMC-7721.
Zhonghua gan zang bing za zhi = Zhonghua ganzangbing zazhi = Chinese journal of hepatology 01/2004; 11(12):712-5.
ABSTRACT: To develop an RNAi approach that specifically targets the core gene sequence of hepatitis B virus by synthesizing short interfering RNA (siRNA) in vivo, and to assess the inhibitory effect of this siRNA on HBV replication.
The eukaryotic expression plasmid pHBV1.3, which contains 1.3-fold-overlength genome of HBV, were cotransfected into HepG2 cells with either the RNAi plasmid pSIHBV/C or unrelated control plasmid pSI. At 24, 48, 72 hours post transfection (p.t.), the levels of HBsAg and HBeAg in the cell culture medium were determined with Abbott MEIA Kits. The expression of intracellular viral proteins was also determined by immunofluorescence staining.
Successfully constructed expressing siRNA vector pSIHBV which targeted the core gene of Hepatitis B virus.The introduction of RNAi plasmid was shown to efficiently and specifically inhibit the synthesis of surface and e antigen of HBV by Abbot MEIA kits, with inhibitory rates at 92%, 85% peaking at 24 hours p.t. Immunofluorescence staining showed that intracellular synthesis of viral antigen was sharply reduced to nearly background levels when the ratio of pSIHBV/C and pHBV1.3 was at 1:20, whereas the control vector did not exhibit any inhibitory effect on the replication and expression of HBV.
Our results demonstrate that the short interfering RNA targeting HBV core gene exerts robust inhibition on HBV viral replication, suggesting that RNAi-based anti-HBV replication strategy may represent a potentially efficacious approach to the clinical management of HBV infection.
Zhonghua yi xue za zhi 09/2003; 83(15):1309-12.
ABSTRACT: To transfer 1.3-fold-overlength genome of HBV expression plasmid into HepG2 cells, and observe the dynamic changes of viral replication as well as expression in the transfected cells.
4.1 kb fragment of HBV genome, derived from pGEM-HBV, was cloned into Hind III site of the eukaryotic expression vector pCDNA3.1 to construct the recombinant plasmid pHBV. Then HepG2 hepatoma, cells were transfected with pHBV, using Lipofectamine2000 transfection reagent. After 24, 48, 72 hours, the levels of HBsAg and HBeAg in the supernatant of HepG2 cells were determined with Abbott MEIA Kit. Intracellular viral DNA and RNA were analyzed by Southern and northern blot hybridization. In addition, viral-specific proteins (HBsAg and HBcAg) were assayed by immunofluorescence staining.
The expression vector pCDNA3.1 was constructed successfully. The levels of HBsAg were 5.36+-0.25, 13.42+-1.24, 7.52+-0.43, and the values of HBeAg were 9.16+-0.32, 22.75+-1.49, 15.96+-1.03 after 24, 48, 72 hours, respectively. All expected HBV replicative intermediates and specific transcripts were verified by Southern and northern blot analysis. The HBsAg-positive cells peaked after 24 hours, and then dropped slowly. HBsAg positive staining scattered in the cytoplasm, whereas HBcAg lied maily in the cytoplasm apart from nuclears.
This recombinant plasmid, which initiates viral replication efficiently in infected cells, is expected as a novel tool for investigating HBV replication in vitro.
Zhonghua gan zang bing za zhi = Zhonghua ganzangbing zazhi = Chinese journal of hepatology 09/2003; 11(8):464-6.
ABSTRACT: To investigate the expression of recombinant IkappaBalphaM (AdIkappaBalphaM) in human hepatocarcinoma cell line HepG(2) and the inhibiting effect to NF-kappaB.
To test the virus titer in 293 cells and the infective efficiency virus titer in HepG(2) cells with GFP and limited dilution method, then to assay the IkappaBalphaM expression of recombinant adenovirus and the alteration after induction of TNF-alpha in 293 cells and HepG(2) by Western blotting, furthermore, to observe NF-kappaB activity in HepG(2) before and after treatment of TNF-alpha by EMSA.
The titer of AdIkappaBalphaM is 2 x 10(8) pfu/L, MOI equals to 20. AdIkappaBalphaM could be expressed stably and efficiently in HepG(2) and will not degrade by induction of TNF-alpha; but IkappaBalpha in the uninfection cell as well as AdIkappaBalpha control was increased at first and then decreased. EMSA demonstrated that the infected cells showed no activation of NF-kappaB before and after the treatment of TNF-alpha, but the cells of uninfected and infected with AdIkappaBalpha appeared excessive activation of NF-kappaB.
AdIkappaBalphaM could be amplified in 293 cell and effectively infect to target cells HepG(2), and could be expressed stably in cells, and wouldn't be degrade with treatment of TNF-alpha, also it can effectually inhibit the excessive activation of NF-kappaB in HepG(2). The result of our research indicates the theoretical value of IkappaBalphaM as a super inhibitor, inhibition activity of NF-kappaB with IkappaBalphaM super-suppressor aided with routine anti-tumor therapy would become an effective method.
Zhonghua yi xue za zhi 08/2003; 83(13):1156-60.