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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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Inhibitory effects of TuD RNA on other family members of the target miRNA. (A) RNA sequences of miR-16, 195 and 497 and homology between them. Sequences shown in blue box are the heptameric seed sequence (2–8 from the 5′ end). Black bars indicated homologous nucleotides. (B) The imperfect pairing between miR-16 and MBSs of the corresponding TuD RNAs. Black dots indicate G-U pairs. (C) TuD RNA expression plasmid vectors were transiently transfected into HCT-116 cells together with the Renilla luciferase miR-16 reporter (open bars) or the untargeted control Renilla luciferase reporter (black bars) as well as the Firefly luciferase reporter as a transfection control. After performing a dual luciferase assay, the expression levels were normalized to the ratio of the activity of miR-16-RL to that of FL in TuD-NC vector-transfected cells and are represented by the mean ± SEM (n = 3).
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Figure 7: Inhibitory effects of TuD RNA on other family members of the target miRNA. (A) RNA sequences of miR-16, 195 and 497 and homology between them. Sequences shown in blue box are the heptameric seed sequence (2–8 from the 5′ end). Black bars indicated homologous nucleotides. (B) The imperfect pairing between miR-16 and MBSs of the corresponding TuD RNAs. Black dots indicate G-U pairs. (C) TuD RNA expression plasmid vectors were transiently transfected into HCT-116 cells together with the Renilla luciferase miR-16 reporter (open bars) or the untargeted control Renilla luciferase reporter (black bars) as well as the Firefly luciferase reporter as a transfection control. After performing a dual luciferase assay, the expression levels were normalized to the ratio of the activity of miR-16-RL to that of FL in TuD-NC vector-transfected cells and are represented by the mean ± SEM (n = 3).

Mentions: To examine the specificity of miRNA more rigidly, we next tested whether the TuD RNA targeted to a miRNA exhibits inhibitory effects to other family members of the miRNA, which share the same core sequence (2–8 bases from the 5′-end of the miRNA). We have chosen the miR-15a/-15b/-16/-195/-424/-497 family and tested in HCT-116 cells, where expression of miR-16 is predominant (Supplementary Figure S6). So we expect the reporter containing the sequence perfectly complementary to miR-16 is mainly reflecting amounts of functional miR-16. In this setting, we induced high-level expression of TuD-miR195-4ntin and TuD-miR497-4ntin, respectively in HCT-116 cells, in which endogenous miR-195 and miR-497 are expressed only marginally (Supplementary Figure S6). The results of reporter assays (Figure 7) indicated the inhibitory activity of TuD-miR195-4ntin is no less than that of TuD-miR16-4ntin, whereas TuD-miR497-4ntin has slightly reduced the inhibitory effect when compared with TuD-miR16-4ntin. Since the reporter expression is not expected to result from the suppression of endogenous miR-195 or miR-497, we concluded that TuD-RNA do not basically discriminate miRNA members that belong to the same miRNA family.Figure 7.


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)

Inhibitory effects of TuD RNA on other family members of the target miRNA. (A) RNA sequences of miR-16, 195 and 497 and homology between them. Sequences shown in blue box are the heptameric seed sequence (2–8 from the 5′ end). Black bars indicated homologous nucleotides. (B) The imperfect pairing between miR-16 and MBSs of the corresponding TuD RNAs. Black dots indicate G-U pairs. (C) TuD RNA expression plasmid vectors were transiently transfected into HCT-116 cells together with the Renilla luciferase miR-16 reporter (open bars) or the untargeted control Renilla luciferase reporter (black bars) as well as the Firefly luciferase reporter as a transfection control. After performing a dual luciferase assay, the expression levels were normalized to the ratio of the activity of miR-16-RL to that of FL in TuD-NC vector-transfected cells and are represented by the mean ± SEM (n = 3).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 7: Inhibitory effects of TuD RNA on other family members of the target miRNA. (A) RNA sequences of miR-16, 195 and 497 and homology between them. Sequences shown in blue box are the heptameric seed sequence (2–8 from the 5′ end). Black bars indicated homologous nucleotides. (B) The imperfect pairing between miR-16 and MBSs of the corresponding TuD RNAs. Black dots indicate G-U pairs. (C) TuD RNA expression plasmid vectors were transiently transfected into HCT-116 cells together with the Renilla luciferase miR-16 reporter (open bars) or the untargeted control Renilla luciferase reporter (black bars) as well as the Firefly luciferase reporter as a transfection control. After performing a dual luciferase assay, the expression levels were normalized to the ratio of the activity of miR-16-RL to that of FL in TuD-NC vector-transfected cells and are represented by the mean ± SEM (n = 3).
Mentions: To examine the specificity of miRNA more rigidly, we next tested whether the TuD RNA targeted to a miRNA exhibits inhibitory effects to other family members of the miRNA, which share the same core sequence (2–8 bases from the 5′-end of the miRNA). We have chosen the miR-15a/-15b/-16/-195/-424/-497 family and tested in HCT-116 cells, where expression of miR-16 is predominant (Supplementary Figure S6). So we expect the reporter containing the sequence perfectly complementary to miR-16 is mainly reflecting amounts of functional miR-16. In this setting, we induced high-level expression of TuD-miR195-4ntin and TuD-miR497-4ntin, respectively in HCT-116 cells, in which endogenous miR-195 and miR-497 are expressed only marginally (Supplementary Figure S6). The results of reporter assays (Figure 7) indicated the inhibitory activity of TuD-miR195-4ntin is no less than that of TuD-miR16-4ntin, whereas TuD-miR497-4ntin has slightly reduced the inhibitory effect when compared with TuD-miR16-4ntin. Since the reporter expression is not expected to result from the suppression of endogenous miR-195 or miR-497, we concluded that TuD-RNA do not basically discriminate miRNA members that belong to the same miRNA family.Figure 7.

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