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RNA Interference inhibits hepatitis B virus of different genotypes in vitro and in vivo.

Zhang YL, Cheng T, Cai YJ, Yuan Q, Liu C, Zhang T, Xia DZ, Li RY, Yang LW, Wang YB, Yeo AE, Shih JW, Zhang J, Xia NS - BMC Microbiol. (2010)

Bottom Line: Small interfering RNA (siRNA) can be a potential new tool for HBV therapy.Given the high heterogeneity of HBV strains and the sensitivity towards sequences changes of siRNA, finding a potent siRNA inhibitor against the conservative site on the HBV genome is essential to ensure a therapeutic application.No unusual cytotoxicity or off-target effects were noted.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Science, Xiamen University, Xiamen, Fujian Province, China.

ABSTRACT

Background: Hepatitis B virus (HBV) infection increases the risk of liver disease and hepatocellular carcinoma. Small interfering RNA (siRNA) can be a potential new tool for HBV therapy. Given the high heterogeneity of HBV strains and the sensitivity towards sequences changes of siRNA, finding a potent siRNA inhibitor against the conservative site on the HBV genome is essential to ensure a therapeutic application.

Results: Forty short hairpin RNA (shRNA) expression plasmids were constructed to target conserved regions among nine HBV genotypes. HBV 1.3-fold genome plasmids carrying various genotypes were co-transfected with shRNA plasmids into either Huh7 cells or mice. The levels of various viral markers were examined to assess the anti-HBV efficacy of siRNA. Four (B245, B376, B1581 and B1789) were found with the ability to potently inhibit HBV RNA, DNA, surface antigen (HBsAg), e antigen (HBeAg) and core antigen (HBcAg) expression in HBV genotypes A, B, C, D and I (a newly identified genotype) in Huh7 cells and in mice. No unusual cytotoxicity or off-target effects were noted.

Conclusions: Such siRNA suggests an alternate way of inhibiting various HBV genotypes in vitro and in vivo, promising advances in the treatment of HBV.

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Related in: MedlinePlus

The expression profile of four major interferon stimulated genes (ISGs) in shRNA plasmids transfected cells. Cytoplasmic RNAs, from Huh7 cells treated with or without IFNα-2a or transfected with either pSUPER vector or shRNA plasmids, were analysed by realtime RT-PCR for IFN stimulated genes STAT1, OAS1, GBP1 and MX1. The values on the figure, plotted as "Relative gene expression level" on the y-axis, were calculated as the mRNA levels of ISGs divided by the GAPDH (control) mRNA level. Student t test was used to assess the difference between shRNA plasmids (including empty pSUPER vector) of transfected cells and non-transfected cells (mock). No significant difference was observed.
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Figure 2: The expression profile of four major interferon stimulated genes (ISGs) in shRNA plasmids transfected cells. Cytoplasmic RNAs, from Huh7 cells treated with or without IFNα-2a or transfected with either pSUPER vector or shRNA plasmids, were analysed by realtime RT-PCR for IFN stimulated genes STAT1, OAS1, GBP1 and MX1. The values on the figure, plotted as "Relative gene expression level" on the y-axis, were calculated as the mRNA levels of ISGs divided by the GAPDH (control) mRNA level. Student t test was used to assess the difference between shRNA plasmids (including empty pSUPER vector) of transfected cells and non-transfected cells (mock). No significant difference was observed.

Mentions: The B245, B376, B1581, and B1789 plasmids were transfected into Huh7 cells to determine cytotoxicity by the WST-8 assay. No significant siRNA-induced cytotoxicity was observed for these siRNA when compared to an empty pSUPER vector (p = 0.66, data not shown). The mRNA levels of four major interferon stimulated genes (STAT1, OAS1, GBP1 and MX1) in transfected cells were measured by quantitative realtime PCR with GAPDH mRNA acting as a control. As shown in Figure 2, between values 1 and 2, logarithmic increases for the IFN-stimulatable mRNAs were only observed in the IFN-treated cells, but not observed in any of the shRNA treated cells vs. untreated cells. From this, it can be concluded that an IFN response is not activated by these anti-HBV siRNAs.


RNA Interference inhibits hepatitis B virus of different genotypes in vitro and in vivo.

Zhang YL, Cheng T, Cai YJ, Yuan Q, Liu C, Zhang T, Xia DZ, Li RY, Yang LW, Wang YB, Yeo AE, Shih JW, Zhang J, Xia NS - BMC Microbiol. (2010)

The expression profile of four major interferon stimulated genes (ISGs) in shRNA plasmids transfected cells. Cytoplasmic RNAs, from Huh7 cells treated with or without IFNα-2a or transfected with either pSUPER vector or shRNA plasmids, were analysed by realtime RT-PCR for IFN stimulated genes STAT1, OAS1, GBP1 and MX1. The values on the figure, plotted as "Relative gene expression level" on the y-axis, were calculated as the mRNA levels of ISGs divided by the GAPDH (control) mRNA level. Student t test was used to assess the difference between shRNA plasmids (including empty pSUPER vector) of transfected cells and non-transfected cells (mock). No significant difference was observed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2927532&req=5

Figure 2: The expression profile of four major interferon stimulated genes (ISGs) in shRNA plasmids transfected cells. Cytoplasmic RNAs, from Huh7 cells treated with or without IFNα-2a or transfected with either pSUPER vector or shRNA plasmids, were analysed by realtime RT-PCR for IFN stimulated genes STAT1, OAS1, GBP1 and MX1. The values on the figure, plotted as "Relative gene expression level" on the y-axis, were calculated as the mRNA levels of ISGs divided by the GAPDH (control) mRNA level. Student t test was used to assess the difference between shRNA plasmids (including empty pSUPER vector) of transfected cells and non-transfected cells (mock). No significant difference was observed.
Mentions: The B245, B376, B1581, and B1789 plasmids were transfected into Huh7 cells to determine cytotoxicity by the WST-8 assay. No significant siRNA-induced cytotoxicity was observed for these siRNA when compared to an empty pSUPER vector (p = 0.66, data not shown). The mRNA levels of four major interferon stimulated genes (STAT1, OAS1, GBP1 and MX1) in transfected cells were measured by quantitative realtime PCR with GAPDH mRNA acting as a control. As shown in Figure 2, between values 1 and 2, logarithmic increases for the IFN-stimulatable mRNAs were only observed in the IFN-treated cells, but not observed in any of the shRNA treated cells vs. untreated cells. From this, it can be concluded that an IFN response is not activated by these anti-HBV siRNAs.

Bottom Line: Small interfering RNA (siRNA) can be a potential new tool for HBV therapy.Given the high heterogeneity of HBV strains and the sensitivity towards sequences changes of siRNA, finding a potent siRNA inhibitor against the conservative site on the HBV genome is essential to ensure a therapeutic application.No unusual cytotoxicity or off-target effects were noted.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Science, Xiamen University, Xiamen, Fujian Province, China.

ABSTRACT

Background: Hepatitis B virus (HBV) infection increases the risk of liver disease and hepatocellular carcinoma. Small interfering RNA (siRNA) can be a potential new tool for HBV therapy. Given the high heterogeneity of HBV strains and the sensitivity towards sequences changes of siRNA, finding a potent siRNA inhibitor against the conservative site on the HBV genome is essential to ensure a therapeutic application.

Results: Forty short hairpin RNA (shRNA) expression plasmids were constructed to target conserved regions among nine HBV genotypes. HBV 1.3-fold genome plasmids carrying various genotypes were co-transfected with shRNA plasmids into either Huh7 cells or mice. The levels of various viral markers were examined to assess the anti-HBV efficacy of siRNA. Four (B245, B376, B1581 and B1789) were found with the ability to potently inhibit HBV RNA, DNA, surface antigen (HBsAg), e antigen (HBeAg) and core antigen (HBcAg) expression in HBV genotypes A, B, C, D and I (a newly identified genotype) in Huh7 cells and in mice. No unusual cytotoxicity or off-target effects were noted.

Conclusions: Such siRNA suggests an alternate way of inhibiting various HBV genotypes in vitro and in vivo, promising advances in the treatment of HBV.

Show MeSH
Related in: MedlinePlus