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Inhibition of core gene of HCV 3a genotype using synthetic and vector derived siRNAs.

Khaliq S, Jahan S, Ijaz B, Ahmad W, Asad S, Pervaiz A, Samreen B, Khan M, Hassan S - Virol. J. (2010)

Bottom Line: Antiviral effects of siRNAs showed upto 80% inhibition of Core gene expression by different siRNAs into Huh-7 cells as compared with Mock transfected and control siRNAs treated cells.For long lasting effect of siRNAs, vector based short hairpin siRNAs (shRNAs) were designed and tested against HCV-3a Core which resulted in a similar pattern of inhibition on RNA and protein expression of HCV Core as synthetic siRNAs.Our results support the possibility of using consensus siRNA and shRNA-based molecular therapy as a promising strategy in effective inhibition of HCV-3a genotype.

View Article: PubMed Central - HTML - PubMed

Affiliation: Applied and Functional Genomics Laboratory, National Center of Excellence in Molecular Biology, University of Punjab, Lahore 53700, Pakistan.

ABSTRACT

Background: Hepatitis C virus (HCV) is a major causative agent of liver associated diseases throughout the world, with genotype 3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current therapy, RNA interference (RNAi) a novel regulatory and powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process represents an alternative option.

Results: The current study was purposed to assess and explore the possibility of RNAi to silence the HCV-3a Core gene expression, which play complex role in regulation of cell growth and host genes expression essential for infectivity and disease progression. To identify the potent siRNA target sites, 5 small interfering RNAs (siRNAs) against Core gene were designed and in vitro transcribed after consensus sequence analysis of different HCV-3a isolates. Antiviral effects of siRNAs showed upto 80% inhibition of Core gene expression by different siRNAs into Huh-7 cells as compared with Mock transfected and control siRNAs treated cells. For long lasting effect of siRNAs, vector based short hairpin siRNAs (shRNAs) were designed and tested against HCV-3a Core which resulted in a similar pattern of inhibition on RNA and protein expression of HCV Core as synthetic siRNAs. Furthermore, the efficacy of cell culture tested siRNA and shRNA, were evaluated for inhibition of HCV replication in HCV infected serum inoculated Huh-7 cells and a significant decrease in HCV viral copy number was observed.

Conclusions: Our results support the possibility of using consensus siRNA and shRNA-based molecular therapy as a promising strategy in effective inhibition of HCV-3a genotype.

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Related in: MedlinePlus

HCV -3a Core specific siRNAs inhibit Core expression. A) Dose dependent silencing effect of synthetic siRNA against Core gene of HCV-3a. Huh-7 cells were transfected with 0.4 μg of constructed HCV Core vector or Mock along with or without 10, 20 and 40 nM of siRNAs for 24 and 48 hrs. Cells were harvested and relative RNA determinations were carried out using semi-quantitative RT-PCR. Gene expression results are given for increasing concentrations of Csi16, Csi27, Csi151, Csi352, and Csi476 siRNAs against HCV-3a Core. Expression levels for Mock-transfected (M), HCV-3a Core expression plasmid (C), scramble siRNA (Sc), 100 bp DNA Ladder (L) and GAPDH are also shown. B) Quantitative Real Time PCR analysis of Core 3a or Mock-treated Huh-7 cells along with or without 40 nM of siRNAs for 24 and 48 hrs in comparison to Mock. Gene expression results from Real Time PCR shows that Csi16, Csi352, and Csi476 siRNAs against HCV-3a Core decrease RNA expression after 24 and 48 hrs transfection. GAPDH was used as internal control. Each independent experiment was performed having triplicate samples. The p values indicate significant differences between the connected groups. Error bars indicate mean S.D, Csi476 verses other siRNA: p* for 24 and p^ for 48 hrs C). Silencing of HCV-3a Core gene by siRNAs using specific antibodies showed reduction at protein expression level. The protein expression levels were determined by western blot analysis after 24 and 48 hrs transfection with Mock (M), HCV-3a Core expression plasmid (C) with and without HCV-3a siRNAs (Csi16, Csi27, Csi151, Csi352, and Csi476) and scramble siRNA (Sc) in Huh-7 cells.
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Figure 1: HCV -3a Core specific siRNAs inhibit Core expression. A) Dose dependent silencing effect of synthetic siRNA against Core gene of HCV-3a. Huh-7 cells were transfected with 0.4 μg of constructed HCV Core vector or Mock along with or without 10, 20 and 40 nM of siRNAs for 24 and 48 hrs. Cells were harvested and relative RNA determinations were carried out using semi-quantitative RT-PCR. Gene expression results are given for increasing concentrations of Csi16, Csi27, Csi151, Csi352, and Csi476 siRNAs against HCV-3a Core. Expression levels for Mock-transfected (M), HCV-3a Core expression plasmid (C), scramble siRNA (Sc), 100 bp DNA Ladder (L) and GAPDH are also shown. B) Quantitative Real Time PCR analysis of Core 3a or Mock-treated Huh-7 cells along with or without 40 nM of siRNAs for 24 and 48 hrs in comparison to Mock. Gene expression results from Real Time PCR shows that Csi16, Csi352, and Csi476 siRNAs against HCV-3a Core decrease RNA expression after 24 and 48 hrs transfection. GAPDH was used as internal control. Each independent experiment was performed having triplicate samples. The p values indicate significant differences between the connected groups. Error bars indicate mean S.D, Csi476 verses other siRNA: p* for 24 and p^ for 48 hrs C). Silencing of HCV-3a Core gene by siRNAs using specific antibodies showed reduction at protein expression level. The protein expression levels were determined by western blot analysis after 24 and 48 hrs transfection with Mock (M), HCV-3a Core expression plasmid (C) with and without HCV-3a siRNAs (Csi16, Csi27, Csi151, Csi352, and Csi476) and scramble siRNA (Sc) in Huh-7 cells.

Mentions: siRNA directed against HCV are expected to successfully block replication cycle since HCV being a RNA virus replicates in the cytoplasm of liver cells without integration into the host genome. To efficiently silence Core gene expression by in-vitro transcribed siRNAs and avoid sequence variants, the most conserved target sequence were chosen after analyzing a consensus sequence of local HCV-3a Core sequences and reference sequences retrieved from GenBank. Negative control siRNA (scrambled siRNA) with the same nucleotide composition as the experimental siRNA which lacks significant sequence homology to the HCV and human genome was designed (Table 1). siRNAs were transfected with pCR3.1/FlagTAG/Core vector into Huh-7 cells to investigate their specificity and expression levels both at mRNA and protein level via semi-quantitative RT-PCR, Real Time PCR and western blotting. To assess the effects of chemically synthesized siRNAs on HCV-3a Core, increasing concentration of siRNAs (Csi16, Csi27, Csi151, Csi352 and Csi476) were introduced into the cells for 24 and 48hrs. All inhibited HCV Core RNA in a dose-dependent manner (10, 20 and 40 nM) examined by semi-quantitative RT-PCR. The inhibitory effect of siRNAs Csi16, Csi352 and Csi476 are stronger even at low concentration, while Csi27 and Csi151 showed more effect after 24 hrs transfection at 40 nM than at 48 hrs post transfection as compared to scrambled siRNA (Figure 1A). These results were further confirmed by Real Time PCR, using primer specific to HCV Core and GAPDH. Based on the relative study, the percentages of HCV mRNA in siRNA (40 nM) co-transfected cells over scramble was calculated, normalizing it with GAPDH. The results of relative quantitative analysis revealed that the mRNA level of HCV Core was decreased to 78% in cells treated with Csi16, 74% with Csi27, 50% with Csi151, 62% with Csi352 and 84% with Csi476 at 24 hrs post transfection while 70% Csi16, 58% with Csi27, 38% with Csi151, 61% with Csi352 and 77% with Csi476 decreased 48 hrs post transfection. The most effective siRNA reaching a maximum inhibition of 60-80% after 24 and 48 hrs transfection were Csi16, Csi27, Csi352 and Csi476 (Figure 1B). Western blot analysis of protein extracts derived from the siRNA transfected cells showed that HCV Core protein expression was reduced in cells co-transfected with Core specific siRNA, but not in the cells transfected with scrambled siRNA 24 and 48 hrs post-transfection. The Csi476 siRNAs was found to be more effective with upto 70% decreased protein expression after 24 hrs while all siRNA inhibited protein expression upto 50-65% after 48 hrs transfection (Figure 1C). These data suggest that synthetic Core siRNA not only has a negative effect on Core mRNA but also it could decrease viral protein production.


Inhibition of core gene of HCV 3a genotype using synthetic and vector derived siRNAs.

Khaliq S, Jahan S, Ijaz B, Ahmad W, Asad S, Pervaiz A, Samreen B, Khan M, Hassan S - Virol. J. (2010)

HCV -3a Core specific siRNAs inhibit Core expression. A) Dose dependent silencing effect of synthetic siRNA against Core gene of HCV-3a. Huh-7 cells were transfected with 0.4 μg of constructed HCV Core vector or Mock along with or without 10, 20 and 40 nM of siRNAs for 24 and 48 hrs. Cells were harvested and relative RNA determinations were carried out using semi-quantitative RT-PCR. Gene expression results are given for increasing concentrations of Csi16, Csi27, Csi151, Csi352, and Csi476 siRNAs against HCV-3a Core. Expression levels for Mock-transfected (M), HCV-3a Core expression plasmid (C), scramble siRNA (Sc), 100 bp DNA Ladder (L) and GAPDH are also shown. B) Quantitative Real Time PCR analysis of Core 3a or Mock-treated Huh-7 cells along with or without 40 nM of siRNAs for 24 and 48 hrs in comparison to Mock. Gene expression results from Real Time PCR shows that Csi16, Csi352, and Csi476 siRNAs against HCV-3a Core decrease RNA expression after 24 and 48 hrs transfection. GAPDH was used as internal control. Each independent experiment was performed having triplicate samples. The p values indicate significant differences between the connected groups. Error bars indicate mean S.D, Csi476 verses other siRNA: p* for 24 and p^ for 48 hrs C). Silencing of HCV-3a Core gene by siRNAs using specific antibodies showed reduction at protein expression level. The protein expression levels were determined by western blot analysis after 24 and 48 hrs transfection with Mock (M), HCV-3a Core expression plasmid (C) with and without HCV-3a siRNAs (Csi16, Csi27, Csi151, Csi352, and Csi476) and scramble siRNA (Sc) in Huh-7 cells.
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Figure 1: HCV -3a Core specific siRNAs inhibit Core expression. A) Dose dependent silencing effect of synthetic siRNA against Core gene of HCV-3a. Huh-7 cells were transfected with 0.4 μg of constructed HCV Core vector or Mock along with or without 10, 20 and 40 nM of siRNAs for 24 and 48 hrs. Cells were harvested and relative RNA determinations were carried out using semi-quantitative RT-PCR. Gene expression results are given for increasing concentrations of Csi16, Csi27, Csi151, Csi352, and Csi476 siRNAs against HCV-3a Core. Expression levels for Mock-transfected (M), HCV-3a Core expression plasmid (C), scramble siRNA (Sc), 100 bp DNA Ladder (L) and GAPDH are also shown. B) Quantitative Real Time PCR analysis of Core 3a or Mock-treated Huh-7 cells along with or without 40 nM of siRNAs for 24 and 48 hrs in comparison to Mock. Gene expression results from Real Time PCR shows that Csi16, Csi352, and Csi476 siRNAs against HCV-3a Core decrease RNA expression after 24 and 48 hrs transfection. GAPDH was used as internal control. Each independent experiment was performed having triplicate samples. The p values indicate significant differences between the connected groups. Error bars indicate mean S.D, Csi476 verses other siRNA: p* for 24 and p^ for 48 hrs C). Silencing of HCV-3a Core gene by siRNAs using specific antibodies showed reduction at protein expression level. The protein expression levels were determined by western blot analysis after 24 and 48 hrs transfection with Mock (M), HCV-3a Core expression plasmid (C) with and without HCV-3a siRNAs (Csi16, Csi27, Csi151, Csi352, and Csi476) and scramble siRNA (Sc) in Huh-7 cells.
Mentions: siRNA directed against HCV are expected to successfully block replication cycle since HCV being a RNA virus replicates in the cytoplasm of liver cells without integration into the host genome. To efficiently silence Core gene expression by in-vitro transcribed siRNAs and avoid sequence variants, the most conserved target sequence were chosen after analyzing a consensus sequence of local HCV-3a Core sequences and reference sequences retrieved from GenBank. Negative control siRNA (scrambled siRNA) with the same nucleotide composition as the experimental siRNA which lacks significant sequence homology to the HCV and human genome was designed (Table 1). siRNAs were transfected with pCR3.1/FlagTAG/Core vector into Huh-7 cells to investigate their specificity and expression levels both at mRNA and protein level via semi-quantitative RT-PCR, Real Time PCR and western blotting. To assess the effects of chemically synthesized siRNAs on HCV-3a Core, increasing concentration of siRNAs (Csi16, Csi27, Csi151, Csi352 and Csi476) were introduced into the cells for 24 and 48hrs. All inhibited HCV Core RNA in a dose-dependent manner (10, 20 and 40 nM) examined by semi-quantitative RT-PCR. The inhibitory effect of siRNAs Csi16, Csi352 and Csi476 are stronger even at low concentration, while Csi27 and Csi151 showed more effect after 24 hrs transfection at 40 nM than at 48 hrs post transfection as compared to scrambled siRNA (Figure 1A). These results were further confirmed by Real Time PCR, using primer specific to HCV Core and GAPDH. Based on the relative study, the percentages of HCV mRNA in siRNA (40 nM) co-transfected cells over scramble was calculated, normalizing it with GAPDH. The results of relative quantitative analysis revealed that the mRNA level of HCV Core was decreased to 78% in cells treated with Csi16, 74% with Csi27, 50% with Csi151, 62% with Csi352 and 84% with Csi476 at 24 hrs post transfection while 70% Csi16, 58% with Csi27, 38% with Csi151, 61% with Csi352 and 77% with Csi476 decreased 48 hrs post transfection. The most effective siRNA reaching a maximum inhibition of 60-80% after 24 and 48 hrs transfection were Csi16, Csi27, Csi352 and Csi476 (Figure 1B). Western blot analysis of protein extracts derived from the siRNA transfected cells showed that HCV Core protein expression was reduced in cells co-transfected with Core specific siRNA, but not in the cells transfected with scrambled siRNA 24 and 48 hrs post-transfection. The Csi476 siRNAs was found to be more effective with upto 70% decreased protein expression after 24 hrs while all siRNA inhibited protein expression upto 50-65% after 48 hrs transfection (Figure 1C). These data suggest that synthetic Core siRNA not only has a negative effect on Core mRNA but also it could decrease viral protein production.

Bottom Line: Antiviral effects of siRNAs showed upto 80% inhibition of Core gene expression by different siRNAs into Huh-7 cells as compared with Mock transfected and control siRNAs treated cells.For long lasting effect of siRNAs, vector based short hairpin siRNAs (shRNAs) were designed and tested against HCV-3a Core which resulted in a similar pattern of inhibition on RNA and protein expression of HCV Core as synthetic siRNAs.Our results support the possibility of using consensus siRNA and shRNA-based molecular therapy as a promising strategy in effective inhibition of HCV-3a genotype.

View Article: PubMed Central - HTML - PubMed

Affiliation: Applied and Functional Genomics Laboratory, National Center of Excellence in Molecular Biology, University of Punjab, Lahore 53700, Pakistan.

ABSTRACT

Background: Hepatitis C virus (HCV) is a major causative agent of liver associated diseases throughout the world, with genotype 3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current therapy, RNA interference (RNAi) a novel regulatory and powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process represents an alternative option.

Results: The current study was purposed to assess and explore the possibility of RNAi to silence the HCV-3a Core gene expression, which play complex role in regulation of cell growth and host genes expression essential for infectivity and disease progression. To identify the potent siRNA target sites, 5 small interfering RNAs (siRNAs) against Core gene were designed and in vitro transcribed after consensus sequence analysis of different HCV-3a isolates. Antiviral effects of siRNAs showed upto 80% inhibition of Core gene expression by different siRNAs into Huh-7 cells as compared with Mock transfected and control siRNAs treated cells. For long lasting effect of siRNAs, vector based short hairpin siRNAs (shRNAs) were designed and tested against HCV-3a Core which resulted in a similar pattern of inhibition on RNA and protein expression of HCV Core as synthetic siRNAs. Furthermore, the efficacy of cell culture tested siRNA and shRNA, were evaluated for inhibition of HCV replication in HCV infected serum inoculated Huh-7 cells and a significant decrease in HCV viral copy number was observed.

Conclusions: Our results support the possibility of using consensus siRNA and shRNA-based molecular therapy as a promising strategy in effective inhibition of HCV-3a genotype.

Show MeSH
Related in: MedlinePlus