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Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells.

Haraguchi T, Ozaki Y, Iba H - Nucleic Acids Res. (2009)

Bottom Line: These inhibitory RNAs were at the same time designed to be expressed in lentiviral vectors and to be transported into the cytoplasm after transcription by RNA polymerase III.We report the optimal conditions that we have established for the design of such RNA decoys (we term these molecules TuD RNAs; tough decoy RNAs).We finally demonstrate that TuD RNAs induce specific and strong biological effects and also show that TuD RNAs achieve the efficient and long-term-suppression of specific miRNAs for over 1 month in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Host-Parasite Interaction, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

ABSTRACT
Whereas the strong and stable suppression of specific microRNA activity would be essential for the functional analysis of these molecules, and also for the development of therapeutic applications, effective inhibitory methods to achieve this have not yet been fully established. In our current study, we tested various RNA decoys which were designed to efficiently expose indigestible complementary RNAs to a specific miRNA molecule. These inhibitory RNAs were at the same time designed to be expressed in lentiviral vectors and to be transported into the cytoplasm after transcription by RNA polymerase III. We report the optimal conditions that we have established for the design of such RNA decoys (we term these molecules TuD RNAs; tough decoy RNAs). We finally demonstrate that TuD RNAs induce specific and strong biological effects and also show that TuD RNAs achieve the efficient and long-term-suppression of specific miRNAs for over 1 month in mammalian cells.

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Biological effects induced by TuD RNA. (A) Inhibition of endogenous miR-21 function induces growth suppression and apoptosis. Cell growth assay was performed after PA-1 cells were transduced with lentivirus vectors expressing either TuD-miR21-4ntin or TuD-NC at an MOI of 10. Cell proliferation was monitored by ATPase based assays and normalized to those of untransduced PA-1 cells on Day 0 and are represented by the mean ± SEM (n = 3). (B) Effects of transduced TuD RNA expression lentivirus vectors upon the expression of endogenous PDCD4 protein, a target of miR-21. PA-1 cells were transduced with the TuD RNA expression lentivirus vectors with an MOI of 10 and total proteins were prepared 20 h after transduction. PDCD4 (top) as well as the β-actin loading control (bottom) were detected by western blotting. (C) Caspase-3 + 7 activation was assayed 48 h after transduction with indicated TuD RNA expression lentivirus with an MOI of 2, 5 and 10. Caspase-3 + 7 activity levels/cell was normalized to that of untransduced PA-1 cells and are represented by the mean ± SEM (n = 3).
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Figure 6: Biological effects induced by TuD RNA. (A) Inhibition of endogenous miR-21 function induces growth suppression and apoptosis. Cell growth assay was performed after PA-1 cells were transduced with lentivirus vectors expressing either TuD-miR21-4ntin or TuD-NC at an MOI of 10. Cell proliferation was monitored by ATPase based assays and normalized to those of untransduced PA-1 cells on Day 0 and are represented by the mean ± SEM (n = 3). (B) Effects of transduced TuD RNA expression lentivirus vectors upon the expression of endogenous PDCD4 protein, a target of miR-21. PA-1 cells were transduced with the TuD RNA expression lentivirus vectors with an MOI of 10 and total proteins were prepared 20 h after transduction. PDCD4 (top) as well as the β-actin loading control (bottom) were detected by western blotting. (C) Caspase-3 + 7 activation was assayed 48 h after transduction with indicated TuD RNA expression lentivirus with an MOI of 2, 5 and 10. Caspase-3 + 7 activity levels/cell was normalized to that of untransduced PA-1 cells and are represented by the mean ± SEM (n = 3).

Mentions: As for biological activity of miR-21, several reports have revealed that it often stimulates cellular proliferation and plays anti-apoptotic roles in several human tumour cell lines (23,24). To test biological effects of the specific suppression of endogenous miR-21 activity, we have transduced lentivirus vectors expressing TuD-miR21-4ntin and TuD-NC into PA-1 cells at a multiplicity of infection (MOI) of 10, where 10 copies of vector provirus in average are integrated into each cell in the transduced culture. We then analysed cellular proliferation by ATPase-based assay (Figure 6A). Cells transduced with TuD-miR21-4ntin have shown only marginal growth (1.6-fold on 4 days after the transduction), whereas TuD-NC transduced cells have grown to more than 5-fold. When an endogenous miR-21 target, PDCD4 protein (24,25) was monitored in the parallel cultures, the expression of PDCD4 was found to be increased by TuD-miR21-4ntin compared with TuD-NC (Figure 6B). When the enzymatic activities of caspase-3 + 7 per cell were determined in similar cultures 2 days after the transduction, for apoptosis by those in TuD-miR21-4ntin transduced cells was about 7- and 3-fold of those in untransduced cells and TuD-NC transduced cells, respectively (Figure 6C), indicating that induction of apoptosis would at least partly explain the reduction of overall growth rate. These cytostatic phenotypes have become detectable even when lentivirus was transduced at an MOI of 2.Figure 6.


Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells.

Haraguchi T, Ozaki Y, Iba H - Nucleic Acids Res. (2009)

Biological effects induced by TuD RNA. (A) Inhibition of endogenous miR-21 function induces growth suppression and apoptosis. Cell growth assay was performed after PA-1 cells were transduced with lentivirus vectors expressing either TuD-miR21-4ntin or TuD-NC at an MOI of 10. Cell proliferation was monitored by ATPase based assays and normalized to those of untransduced PA-1 cells on Day 0 and are represented by the mean ± SEM (n = 3). (B) Effects of transduced TuD RNA expression lentivirus vectors upon the expression of endogenous PDCD4 protein, a target of miR-21. PA-1 cells were transduced with the TuD RNA expression lentivirus vectors with an MOI of 10 and total proteins were prepared 20 h after transduction. PDCD4 (top) as well as the β-actin loading control (bottom) were detected by western blotting. (C) Caspase-3 + 7 activation was assayed 48 h after transduction with indicated TuD RNA expression lentivirus with an MOI of 2, 5 and 10. Caspase-3 + 7 activity levels/cell was normalized to that of untransduced PA-1 cells and are represented by the mean ± SEM (n = 3).
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Related In: Results  -  Collection

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Figure 6: Biological effects induced by TuD RNA. (A) Inhibition of endogenous miR-21 function induces growth suppression and apoptosis. Cell growth assay was performed after PA-1 cells were transduced with lentivirus vectors expressing either TuD-miR21-4ntin or TuD-NC at an MOI of 10. Cell proliferation was monitored by ATPase based assays and normalized to those of untransduced PA-1 cells on Day 0 and are represented by the mean ± SEM (n = 3). (B) Effects of transduced TuD RNA expression lentivirus vectors upon the expression of endogenous PDCD4 protein, a target of miR-21. PA-1 cells were transduced with the TuD RNA expression lentivirus vectors with an MOI of 10 and total proteins were prepared 20 h after transduction. PDCD4 (top) as well as the β-actin loading control (bottom) were detected by western blotting. (C) Caspase-3 + 7 activation was assayed 48 h after transduction with indicated TuD RNA expression lentivirus with an MOI of 2, 5 and 10. Caspase-3 + 7 activity levels/cell was normalized to that of untransduced PA-1 cells and are represented by the mean ± SEM (n = 3).
Mentions: As for biological activity of miR-21, several reports have revealed that it often stimulates cellular proliferation and plays anti-apoptotic roles in several human tumour cell lines (23,24). To test biological effects of the specific suppression of endogenous miR-21 activity, we have transduced lentivirus vectors expressing TuD-miR21-4ntin and TuD-NC into PA-1 cells at a multiplicity of infection (MOI) of 10, where 10 copies of vector provirus in average are integrated into each cell in the transduced culture. We then analysed cellular proliferation by ATPase-based assay (Figure 6A). Cells transduced with TuD-miR21-4ntin have shown only marginal growth (1.6-fold on 4 days after the transduction), whereas TuD-NC transduced cells have grown to more than 5-fold. When an endogenous miR-21 target, PDCD4 protein (24,25) was monitored in the parallel cultures, the expression of PDCD4 was found to be increased by TuD-miR21-4ntin compared with TuD-NC (Figure 6B). When the enzymatic activities of caspase-3 + 7 per cell were determined in similar cultures 2 days after the transduction, for apoptosis by those in TuD-miR21-4ntin transduced cells was about 7- and 3-fold of those in untransduced cells and TuD-NC transduced cells, respectively (Figure 6C), indicating that induction of apoptosis would at least partly explain the reduction of overall growth rate. These cytostatic phenotypes have become detectable even when lentivirus was transduced at an MOI of 2.Figure 6.

Bottom Line: These inhibitory RNAs were at the same time designed to be expressed in lentiviral vectors and to be transported into the cytoplasm after transcription by RNA polymerase III.We report the optimal conditions that we have established for the design of such RNA decoys (we term these molecules TuD RNAs; tough decoy RNAs).We finally demonstrate that TuD RNAs induce specific and strong biological effects and also show that TuD RNAs achieve the efficient and long-term-suppression of specific miRNAs for over 1 month in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Host-Parasite Interaction, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

ABSTRACT
Whereas the strong and stable suppression of specific microRNA activity would be essential for the functional analysis of these molecules, and also for the development of therapeutic applications, effective inhibitory methods to achieve this have not yet been fully established. In our current study, we tested various RNA decoys which were designed to efficiently expose indigestible complementary RNAs to a specific miRNA molecule. These inhibitory RNAs were at the same time designed to be expressed in lentiviral vectors and to be transported into the cytoplasm after transcription by RNA polymerase III. We report the optimal conditions that we have established for the design of such RNA decoys (we term these molecules TuD RNAs; tough decoy RNAs). We finally demonstrate that TuD RNAs induce specific and strong biological effects and also show that TuD RNAs achieve the efficient and long-term-suppression of specific miRNAs for over 1 month in mammalian cells.

Show MeSH
Related in: MedlinePlus