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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 endogenous miR-21 activity. TuD RNA expression plasmid vectors or LNA/DNA antisense oligonucleotides were transiently transfected into PA-1 (A) or HCT-116 (B) cells together with the Renilla luciferase miR-21 reporter (miR-21-RL) (open bars) or the untargeted control Renilla luciferase reporter (UT-RL) (black bars) as well as the Firefly luciferase reporter (FL) as a transfection control. After performing a dual luciferase assay, the expression levels were normalized to the ratio of the activity of miR-21-RL to that of FL in TuD-NC vector-transfected PA-1 (A) or HCT-116 (B) cells and are represented by the mean ± SEM (n = 3). (C) Comparison of the inhibitory effects of TuD RNA and a conventional miRNA inhibitory vector. A dual luciferase assay was performed 72 h after transfection in HCT-116 cells. The ratio of miR-21-RL/FL to UT-RL/FL are represented by the mean ± SEM (n = 3) as the expression levels. (D) The levels of mature miR-21 in RNA from HCT-116 cells transfected with TuD-miR21 expression vectors determined by quantitative real time RT-PCR. The miR-21 expression levels were normalized to those of HCT-116 cells transfected with TuD-NC expression vector and are represented by the mean ± SEM (n = 3). U6 snRNA was served as an endogenous control. (E) The levels of pre- and mature miR-21 in the same RNA samples as (D) determined by northern blotting. Y4 scRNA was served as a loading control.
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Figure 4: Inhibitory effects of TuD RNA on endogenous miR-21 activity. TuD RNA expression plasmid vectors or LNA/DNA antisense oligonucleotides were transiently transfected into PA-1 (A) or HCT-116 (B) cells together with the Renilla luciferase miR-21 reporter (miR-21-RL) (open bars) or the untargeted control Renilla luciferase reporter (UT-RL) (black bars) as well as the Firefly luciferase reporter (FL) as a transfection control. After performing a dual luciferase assay, the expression levels were normalized to the ratio of the activity of miR-21-RL to that of FL in TuD-NC vector-transfected PA-1 (A) or HCT-116 (B) cells and are represented by the mean ± SEM (n = 3). (C) Comparison of the inhibitory effects of TuD RNA and a conventional miRNA inhibitory vector. A dual luciferase assay was performed 72 h after transfection in HCT-116 cells. The ratio of miR-21-RL/FL to UT-RL/FL are represented by the mean ± SEM (n = 3) as the expression levels. (D) The levels of mature miR-21 in RNA from HCT-116 cells transfected with TuD-miR21 expression vectors determined by quantitative real time RT-PCR. The miR-21 expression levels were normalized to those of HCT-116 cells transfected with TuD-NC expression vector and are represented by the mean ± SEM (n = 3). U6 snRNA was served as an endogenous control. (E) The levels of pre- and mature miR-21 in the same RNA samples as (D) determined by northern blotting. Y4 scRNA was served as a loading control.

Mentions: To examine whether the inhibitory functions of TuD RNA had broader applicability, we next performed several experiments using a different miRNA, endogenous miR-21, as the target and employed different assay systems whereby a comparison with traditional synthetic miRNA inhibitors was possible. We designed two TuD-miR 21s, TuD-miR21-4ntin and TuD-miR21-pf, via the same principles and procedures used to generate TuD-miR 140-3p-4ntin, and TuD-miR140-3p-pf, respectively (Supplementary Figure S4). We next tested whether these decoys would suppress endogenous miR-21 activity in PA-1 cells and transiently cotransfected them using DNA-based vectors together with Dual Luciferase Reporters (DLR) i.e. the Renilla luciferase reporter with or without the insertion of a 22-bp DNA sequence fully complementary to the mature miR-21 into its 3′-UTR as well as the Firefly luciferase reporter as a transfection control (Supplementary Figure S5A–C). As shown in Figure 4A, PA-1 cell populations expressing TuD-miR21-4ntin showed a 25-fold higher expression of miR-21 reporter Renilla luciferase activity, normalized with Firefly luciferase activity, compared with a vector for negative control (TuD-NC) (Supplementary Figure S4). TuD-miR21-pf showed lower inhibitory effects but still induced a significant recovery corresponding to about a 14-fold activation. Importantly, the levels of recovered reporter activity were close to those of the control reporter without the miR-21 target sequence. As expected, this control reporter activity was found to be very constant and independent of the decoy RNAs introduced. Transfection of locked nucleic acid antisense oligonucleotides (LNA/DNA), LNA/DNA-miR21 showed a 2-fold higher expression of the miR-21 target reporter compared with LNA/DNA-scramble transfected PA-1 cells (Figure 4A), showing that TuD RNAs have a much greater inhibitory potency towards miRNA than conventional synthetic reagents under these transfection conditions.Figure 4.


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 endogenous miR-21 activity. TuD RNA expression plasmid vectors or LNA/DNA antisense oligonucleotides were transiently transfected into PA-1 (A) or HCT-116 (B) cells together with the Renilla luciferase miR-21 reporter (miR-21-RL) (open bars) or the untargeted control Renilla luciferase reporter (UT-RL) (black bars) as well as the Firefly luciferase reporter (FL) as a transfection control. After performing a dual luciferase assay, the expression levels were normalized to the ratio of the activity of miR-21-RL to that of FL in TuD-NC vector-transfected PA-1 (A) or HCT-116 (B) cells and are represented by the mean ± SEM (n = 3). (C) Comparison of the inhibitory effects of TuD RNA and a conventional miRNA inhibitory vector. A dual luciferase assay was performed 72 h after transfection in HCT-116 cells. The ratio of miR-21-RL/FL to UT-RL/FL are represented by the mean ± SEM (n = 3) as the expression levels. (D) The levels of mature miR-21 in RNA from HCT-116 cells transfected with TuD-miR21 expression vectors determined by quantitative real time RT-PCR. The miR-21 expression levels were normalized to those of HCT-116 cells transfected with TuD-NC expression vector and are represented by the mean ± SEM (n = 3). U6 snRNA was served as an endogenous control. (E) The levels of pre- and mature miR-21 in the same RNA samples as (D) determined by northern blotting. Y4 scRNA was served as a loading control.
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Figure 4: Inhibitory effects of TuD RNA on endogenous miR-21 activity. TuD RNA expression plasmid vectors or LNA/DNA antisense oligonucleotides were transiently transfected into PA-1 (A) or HCT-116 (B) cells together with the Renilla luciferase miR-21 reporter (miR-21-RL) (open bars) or the untargeted control Renilla luciferase reporter (UT-RL) (black bars) as well as the Firefly luciferase reporter (FL) as a transfection control. After performing a dual luciferase assay, the expression levels were normalized to the ratio of the activity of miR-21-RL to that of FL in TuD-NC vector-transfected PA-1 (A) or HCT-116 (B) cells and are represented by the mean ± SEM (n = 3). (C) Comparison of the inhibitory effects of TuD RNA and a conventional miRNA inhibitory vector. A dual luciferase assay was performed 72 h after transfection in HCT-116 cells. The ratio of miR-21-RL/FL to UT-RL/FL are represented by the mean ± SEM (n = 3) as the expression levels. (D) The levels of mature miR-21 in RNA from HCT-116 cells transfected with TuD-miR21 expression vectors determined by quantitative real time RT-PCR. The miR-21 expression levels were normalized to those of HCT-116 cells transfected with TuD-NC expression vector and are represented by the mean ± SEM (n = 3). U6 snRNA was served as an endogenous control. (E) The levels of pre- and mature miR-21 in the same RNA samples as (D) determined by northern blotting. Y4 scRNA was served as a loading control.
Mentions: To examine whether the inhibitory functions of TuD RNA had broader applicability, we next performed several experiments using a different miRNA, endogenous miR-21, as the target and employed different assay systems whereby a comparison with traditional synthetic miRNA inhibitors was possible. We designed two TuD-miR 21s, TuD-miR21-4ntin and TuD-miR21-pf, via the same principles and procedures used to generate TuD-miR 140-3p-4ntin, and TuD-miR140-3p-pf, respectively (Supplementary Figure S4). We next tested whether these decoys would suppress endogenous miR-21 activity in PA-1 cells and transiently cotransfected them using DNA-based vectors together with Dual Luciferase Reporters (DLR) i.e. the Renilla luciferase reporter with or without the insertion of a 22-bp DNA sequence fully complementary to the mature miR-21 into its 3′-UTR as well as the Firefly luciferase reporter as a transfection control (Supplementary Figure S5A–C). As shown in Figure 4A, PA-1 cell populations expressing TuD-miR21-4ntin showed a 25-fold higher expression of miR-21 reporter Renilla luciferase activity, normalized with Firefly luciferase activity, compared with a vector for negative control (TuD-NC) (Supplementary Figure S4). TuD-miR21-pf showed lower inhibitory effects but still induced a significant recovery corresponding to about a 14-fold activation. Importantly, the levels of recovered reporter activity were close to those of the control reporter without the miR-21 target sequence. As expected, this control reporter activity was found to be very constant and independent of the decoy RNAs introduced. Transfection of locked nucleic acid antisense oligonucleotides (LNA/DNA), LNA/DNA-miR21 showed a 2-fold higher expression of the miR-21 target reporter compared with LNA/DNA-scramble transfected PA-1 cells (Figure 4A), showing that TuD RNAs have a much greater inhibitory potency towards miRNA than conventional synthetic reagents under these transfection conditions.Figure 4.

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