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Down-regulation of IRES containing 5'UTR of HCV genotype 3a using siRNAs.

Khaliq S, Jahan S, Pervaiz A, Ali Ashfaq U, Hassan S - Virol. J. (2011)

Bottom Line: For this analysis full length HCV 5'UTR of HCV-3a (pCR3.1/5'UTR) was tagged with GFP protein for in vitro analysis in Huh-7 cells. siRNA targeting 5'UTR were designed, and tested against constructed vector in Huh-7 cell line both at RNA and Protein levels.Furthermore, the effect of these siRNAs was confirmed in HCV-3a serum infected Huh-7 cell line.The potential of siRNAs specificity to inhibit HCV-3a replication in serum-infected Huh-7 cells was also investigated; upon treatment with siRNAs a significant decrease in HCV viral copy number and protein expression was observed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Applied and Functional Genomics Lab, Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan. sabahat711@yahoo.com

ABSTRACT

Background: Hepatitis C virus (HCV) is a major causative agent of liver associated diseases leading to the development of hepatocellular carcinoma (HCC) all over the world and genotype-3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current chemotherapy of interferon-α (IFN-α) and ribavirin against HCV infection alternative options are desperately needed out of which the recently discovered RNAi represent a powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process to silence virus infection or replication. HCV translation is mediated by a highly conserved internal ribosome entry site (IRES) within the 5'UTR region making it a relevant target for new drug development.

Materials and methods: The present study was proposed to assess and explore the possibility of HCV silencing using siRNA targeting 5'UTR. For this analysis full length HCV 5'UTR of HCV-3a (pCR3.1/5'UTR) was tagged with GFP protein for in vitro analysis in Huh-7 cells. siRNA targeting 5'UTR were designed, and tested against constructed vector in Huh-7 cell line both at RNA and Protein levels. Furthermore, the effect of these siRNAs was confirmed in HCV-3a serum infected Huh-7 cell line.

Results: The expression of 5'UTR-GFP was dramatically reduced both at mRNA and protein levels as compared with Mock transfected and control siRNAs treated cells using siRNAs against IRES of HCV-3a genotype. The potential of siRNAs specificity to inhibit HCV-3a replication in serum-infected Huh-7 cells was also investigated; upon treatment with siRNAs a significant decrease in HCV viral copy number and protein expression was observed.

Conclusions: Overall, the present work of siRNAs against HCV 5'UTR inhibits HCV-3a expression and represents effective future therapeutic opportunities against HCV-3a genotype.

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Related in: MedlinePlus

HCV 3a 5'UTR specific siRNAs inhibit mRNA expression. A) Huh-7 cells were transfected with 0.4 μg of constructed HCV vector or mock-treated along with or without 10 nM, 20 nM and 40 nM of siRNAs for 24 and 48 hrs. Cells were harvested and relative RNA determinations were carried out using semi-quantitative PCR. Gene expression results from semi-quantitative PCR are given for increasing concentrations of Usi170, Usi212 and Usi272 siRNAs against HCV 3a 5'UTR. Expression levels for mock-transfected (M), HCV 3a 5'UTR expression plasmid (U), scramble siRNA (Sc), 100 bp DNA Ladder (L) and GAPDH are also shown. B) Huh-7 cells were transfected pCR3.1/GFP/5'UTR vector or mock-treated along with or without 40 nM of siRNAs for 24 and 48 hrs. Total cellular RNA extracted, after 24 and 48 hrs post transfection, was quantified by Real Time PCR using gene specific primers in comparison to Mock. Gene expression results from Real Time PCR shows that Usi170 and Usi272 siRNAs against HCV 3a 5'UTR decrease RNA expression both after 24 and 48 hrs post transfection. GAPDH was used as internal control. Three independent experiments were performed having triplicate samples. Error bars indicate mean S.D, p < 0.01.
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Figure 1: HCV 3a 5'UTR specific siRNAs inhibit mRNA expression. A) Huh-7 cells were transfected with 0.4 μg of constructed HCV vector or mock-treated along with or without 10 nM, 20 nM and 40 nM of siRNAs for 24 and 48 hrs. Cells were harvested and relative RNA determinations were carried out using semi-quantitative PCR. Gene expression results from semi-quantitative PCR are given for increasing concentrations of Usi170, Usi212 and Usi272 siRNAs against HCV 3a 5'UTR. Expression levels for mock-transfected (M), HCV 3a 5'UTR expression plasmid (U), scramble siRNA (Sc), 100 bp DNA Ladder (L) and GAPDH are also shown. B) Huh-7 cells were transfected pCR3.1/GFP/5'UTR vector or mock-treated along with or without 40 nM of siRNAs for 24 and 48 hrs. Total cellular RNA extracted, after 24 and 48 hrs post transfection, was quantified by Real Time PCR using gene specific primers in comparison to Mock. Gene expression results from Real Time PCR shows that Usi170 and Usi272 siRNAs against HCV 3a 5'UTR decrease RNA expression both after 24 and 48 hrs post transfection. GAPDH was used as internal control. Three independent experiments were performed having triplicate samples. Error bars indicate mean S.D, p < 0.01.

Mentions: Due to highly conserved region of HCV is indispensible for both RNA translation and replication 5'UTR has been a focus of antiviral research, therefore in the present study we designed and tested siRNAs targeting 5'UTR of HCV-3a to evaluate their antiviral activity against important domains for translation initiation. To test the HCV 3a directed siRNAs for their ability to suppress the well conserved HCV IRES (5'UTR) mediated translation, three siRNAs were designed to trigger RNAi and co-transfected with constructed vector into Huh-7 cells in different concentrations. Huh-7 cells were transfected with pCR3.1/GFP/5'UTR, that expresses GFP under the control of HCV 5'UTR, with and without 5'UTR specific siRNAs. All siRNAs inhibited the expression of GFP gene in a dose dependent manner (10, 20 and 40 nM) compared with control siRNA. The inhibitory effect of Usi170 siRNA against 5'UTR is stronger at 24 hrs and 48 hrs post transfection even at low concentration, while Usi212 and Usi272 shows more effect after 24 and 48 hrs transfection at 40 nM compared with scramble siRNA which showed no significant change in expression of transfected vector to control cells. In all the siRNA screening experiments, treatment with synthetic and control siRNA did not affect the levels of cellular gene GAPDH expression (Figure 1A). The results of relative quantitative analysis revealed that the transcript levels of the HCV 5'UTR were decreased to 67% in treated cells with Usi170, 71% with Usi212 and 62% with Usi272 at 24 hrs post-transfection, while 65% with Usi170, 48% with Usi212 and 36.7% with Usi272 at 48 hrs post-transfection. A maximum inhibition of 60-70% was observed with Usi170. GAPDH transcript levels showed no change in non-transfected or transfected cells (Figure 1B).


Down-regulation of IRES containing 5'UTR of HCV genotype 3a using siRNAs.

Khaliq S, Jahan S, Pervaiz A, Ali Ashfaq U, Hassan S - Virol. J. (2011)

HCV 3a 5'UTR specific siRNAs inhibit mRNA expression. A) Huh-7 cells were transfected with 0.4 μg of constructed HCV vector or mock-treated along with or without 10 nM, 20 nM and 40 nM of siRNAs for 24 and 48 hrs. Cells were harvested and relative RNA determinations were carried out using semi-quantitative PCR. Gene expression results from semi-quantitative PCR are given for increasing concentrations of Usi170, Usi212 and Usi272 siRNAs against HCV 3a 5'UTR. Expression levels for mock-transfected (M), HCV 3a 5'UTR expression plasmid (U), scramble siRNA (Sc), 100 bp DNA Ladder (L) and GAPDH are also shown. B) Huh-7 cells were transfected pCR3.1/GFP/5'UTR vector or mock-treated along with or without 40 nM of siRNAs for 24 and 48 hrs. Total cellular RNA extracted, after 24 and 48 hrs post transfection, was quantified by Real Time PCR using gene specific primers in comparison to Mock. Gene expression results from Real Time PCR shows that Usi170 and Usi272 siRNAs against HCV 3a 5'UTR decrease RNA expression both after 24 and 48 hrs post transfection. GAPDH was used as internal control. Three independent experiments were performed having triplicate samples. Error bars indicate mean S.D, p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3116492&req=5

Figure 1: HCV 3a 5'UTR specific siRNAs inhibit mRNA expression. A) Huh-7 cells were transfected with 0.4 μg of constructed HCV vector or mock-treated along with or without 10 nM, 20 nM and 40 nM of siRNAs for 24 and 48 hrs. Cells were harvested and relative RNA determinations were carried out using semi-quantitative PCR. Gene expression results from semi-quantitative PCR are given for increasing concentrations of Usi170, Usi212 and Usi272 siRNAs against HCV 3a 5'UTR. Expression levels for mock-transfected (M), HCV 3a 5'UTR expression plasmid (U), scramble siRNA (Sc), 100 bp DNA Ladder (L) and GAPDH are also shown. B) Huh-7 cells were transfected pCR3.1/GFP/5'UTR vector or mock-treated along with or without 40 nM of siRNAs for 24 and 48 hrs. Total cellular RNA extracted, after 24 and 48 hrs post transfection, was quantified by Real Time PCR using gene specific primers in comparison to Mock. Gene expression results from Real Time PCR shows that Usi170 and Usi272 siRNAs against HCV 3a 5'UTR decrease RNA expression both after 24 and 48 hrs post transfection. GAPDH was used as internal control. Three independent experiments were performed having triplicate samples. Error bars indicate mean S.D, p < 0.01.
Mentions: Due to highly conserved region of HCV is indispensible for both RNA translation and replication 5'UTR has been a focus of antiviral research, therefore in the present study we designed and tested siRNAs targeting 5'UTR of HCV-3a to evaluate their antiviral activity against important domains for translation initiation. To test the HCV 3a directed siRNAs for their ability to suppress the well conserved HCV IRES (5'UTR) mediated translation, three siRNAs were designed to trigger RNAi and co-transfected with constructed vector into Huh-7 cells in different concentrations. Huh-7 cells were transfected with pCR3.1/GFP/5'UTR, that expresses GFP under the control of HCV 5'UTR, with and without 5'UTR specific siRNAs. All siRNAs inhibited the expression of GFP gene in a dose dependent manner (10, 20 and 40 nM) compared with control siRNA. The inhibitory effect of Usi170 siRNA against 5'UTR is stronger at 24 hrs and 48 hrs post transfection even at low concentration, while Usi212 and Usi272 shows more effect after 24 and 48 hrs transfection at 40 nM compared with scramble siRNA which showed no significant change in expression of transfected vector to control cells. In all the siRNA screening experiments, treatment with synthetic and control siRNA did not affect the levels of cellular gene GAPDH expression (Figure 1A). The results of relative quantitative analysis revealed that the transcript levels of the HCV 5'UTR were decreased to 67% in treated cells with Usi170, 71% with Usi212 and 62% with Usi272 at 24 hrs post-transfection, while 65% with Usi170, 48% with Usi212 and 36.7% with Usi272 at 48 hrs post-transfection. A maximum inhibition of 60-70% was observed with Usi170. GAPDH transcript levels showed no change in non-transfected or transfected cells (Figure 1B).

Bottom Line: For this analysis full length HCV 5'UTR of HCV-3a (pCR3.1/5'UTR) was tagged with GFP protein for in vitro analysis in Huh-7 cells. siRNA targeting 5'UTR were designed, and tested against constructed vector in Huh-7 cell line both at RNA and Protein levels.Furthermore, the effect of these siRNAs was confirmed in HCV-3a serum infected Huh-7 cell line.The potential of siRNAs specificity to inhibit HCV-3a replication in serum-infected Huh-7 cells was also investigated; upon treatment with siRNAs a significant decrease in HCV viral copy number and protein expression was observed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Applied and Functional Genomics Lab, Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan. sabahat711@yahoo.com

ABSTRACT

Background: Hepatitis C virus (HCV) is a major causative agent of liver associated diseases leading to the development of hepatocellular carcinoma (HCC) all over the world and genotype-3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current chemotherapy of interferon-α (IFN-α) and ribavirin against HCV infection alternative options are desperately needed out of which the recently discovered RNAi represent a powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process to silence virus infection or replication. HCV translation is mediated by a highly conserved internal ribosome entry site (IRES) within the 5'UTR region making it a relevant target for new drug development.

Materials and methods: The present study was proposed to assess and explore the possibility of HCV silencing using siRNA targeting 5'UTR. For this analysis full length HCV 5'UTR of HCV-3a (pCR3.1/5'UTR) was tagged with GFP protein for in vitro analysis in Huh-7 cells. siRNA targeting 5'UTR were designed, and tested against constructed vector in Huh-7 cell line both at RNA and Protein levels. Furthermore, the effect of these siRNAs was confirmed in HCV-3a serum infected Huh-7 cell line.

Results: The expression of 5'UTR-GFP was dramatically reduced both at mRNA and protein levels as compared with Mock transfected and control siRNAs treated cells using siRNAs against IRES of HCV-3a genotype. The potential of siRNAs specificity to inhibit HCV-3a replication in serum-infected Huh-7 cells was also investigated; upon treatment with siRNAs a significant decrease in HCV viral copy number and protein expression was observed.

Conclusions: Overall, the present work of siRNAs against HCV 5'UTR inhibits HCV-3a expression and represents effective future therapeutic opportunities against HCV-3a genotype.

Show MeSH
Related in: MedlinePlus