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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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Related in: MedlinePlus

Detection of TuD RNA molecules by northern blotting. TuD RNA expression plasmid vectors were transfected into PA-1 (A) or HCT-116 (B) cells and TuD-miR21-4ntin and TuD-miR21-pf were detected by northern blotting. The positions of Y4 scRNA (93 nt) and ACA1 snoRNA (130 nt) are indicated by black and open triangles, respectively. Y4 scRNA was served as a loading control.
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Figure 5: Detection of TuD RNA molecules by northern blotting. TuD RNA expression plasmid vectors were transfected into PA-1 (A) or HCT-116 (B) cells and TuD-miR21-4ntin and TuD-miR21-pf were detected by northern blotting. The positions of Y4 scRNA (93 nt) and ACA1 snoRNA (130 nt) are indicated by black and open triangles, respectively. Y4 scRNA was served as a loading control.

Mentions: We performed similar experiments using HCT-116 cells, which reportedly have higher levels of endogenous miR-21 than PA-1 cells (18,22). We obtained very similar results in this cell system, except that the inhibitory effects of TuD-miR21-pf were dramatically lower than in PA-1 cells (Figure 4B). We have hypothesized that large amounts of endogenous miR-21 would efficiently cleave the TuD-miR21-pf molecule thus significantly reducing its concentration as compared with TuD-miR21-4ntin. To test this hypothesis, we determined the expression level of TuD-miR21-4ntin and TuD-miR21-pf in PA-1 and HCT-116 cells transfected in similar procedures to those shown in Figure 4A and B, except that the reporter plasmids were not included in the transfection. In PA-1 cells, both TuD-miR21-4ntin and TuD-miR21-pf were detected as strong bands (Figure 5A). In HCT-116 cells, TuD-miR21-4ntin was also highly expressed, whereas the expression level of TuD-miR21-pf was very low (Figure 5B), consistent with our hypothesis that TuD-miR21-4ntin is stable, whereas TuD-miR21-pf is susceptible to digestion, especially when cellular concentrations of miR-21 are high. Considering these results, TuD-miRX-4ntin should be used rather than TuD-miRX-pf, which does not have the expected activity as Tough Decoy.Figure 5.


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)

Detection of TuD RNA molecules by northern blotting. TuD RNA expression plasmid vectors were transfected into PA-1 (A) or HCT-116 (B) cells and TuD-miR21-4ntin and TuD-miR21-pf were detected by northern blotting. The positions of Y4 scRNA (93 nt) and ACA1 snoRNA (130 nt) are indicated by black and open triangles, respectively. Y4 scRNA was served as a loading control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665227&req=5

Figure 5: Detection of TuD RNA molecules by northern blotting. TuD RNA expression plasmid vectors were transfected into PA-1 (A) or HCT-116 (B) cells and TuD-miR21-4ntin and TuD-miR21-pf were detected by northern blotting. The positions of Y4 scRNA (93 nt) and ACA1 snoRNA (130 nt) are indicated by black and open triangles, respectively. Y4 scRNA was served as a loading control.
Mentions: We performed similar experiments using HCT-116 cells, which reportedly have higher levels of endogenous miR-21 than PA-1 cells (18,22). We obtained very similar results in this cell system, except that the inhibitory effects of TuD-miR21-pf were dramatically lower than in PA-1 cells (Figure 4B). We have hypothesized that large amounts of endogenous miR-21 would efficiently cleave the TuD-miR21-pf molecule thus significantly reducing its concentration as compared with TuD-miR21-4ntin. To test this hypothesis, we determined the expression level of TuD-miR21-4ntin and TuD-miR21-pf in PA-1 and HCT-116 cells transfected in similar procedures to those shown in Figure 4A and B, except that the reporter plasmids were not included in the transfection. In PA-1 cells, both TuD-miR21-4ntin and TuD-miR21-pf were detected as strong bands (Figure 5A). In HCT-116 cells, TuD-miR21-4ntin was also highly expressed, whereas the expression level of TuD-miR21-pf was very low (Figure 5B), consistent with our hypothesis that TuD-miR21-4ntin is stable, whereas TuD-miR21-pf is susceptible to digestion, especially when cellular concentrations of miR-21 are high. Considering these results, TuD-miRX-4ntin should be used rather than TuD-miRX-pf, which does not have the expected activity as Tough Decoy.Figure 5.

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