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Decoy oligonucleotide rescues IGF1R expression from MicroRNA-223 suppression.

Wu LH, Cai QQ, Dong YW, Wang R, He BM, Qi B, Xu CJ, Wu XZ - PLoS ONE (2013)

Bottom Line: All decoys had no effect on PAXIP1 which was not targeted by miR-223.The decoy 1 that was based on the sequence of IGF1R 3'UTR rescued the expression of IGF1R more significantly than other decoy nucleotides except the antisense decoy 4.However decoy 1 failed to recover Sp1, CDC27 and FBXW7 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Children's Health Care, Yu Ying Children's Hospital, Wenzhou Medical College, Wenzhou, China.

ABSTRACT
A mature miRNA generally suppresses hundreds of mRNA targets. To evaluate the selective effect of synthetic oligonucleotide decoys on hsa-miR-223 activity, reporters containing 3' untranslated regions (UTR) of IGF1R, FOXO1, POLR3G, FOXO3, CDC27, FBXW7 and PAXIP1 mRNAs were constructed for the luciferase assay. The oligonucleotide decoys were designed and synthesized according to mature miR-223 sequence and its target mRNA sequence. Quantitative RT-PCR & western analysis were used to measure miR-223-targeted mRNA expression, Interestingly, apart from the antisense oligonucleotide, decoy nucleotides which were complementary to the 5', central or 3' region of mature miR-223 suppressed miR-223 targeting the 3'UTR of IGF1R, FOXO1, FOXO3, CDC27, POLR3G, and FBXW7 mRNAs and rescued the expression of these genes to varying degrees from miR-223 suppression at both mRNA and protein levels. All decoys had no effect on PAXIP1 which was not targeted by miR-223. The decoy 1 that was based on the sequence of IGF1R 3'UTR rescued the expression of IGF1R more significantly than other decoy nucleotides except the antisense decoy 4. Decoy 1 also rescued the expression of FOXO3 and POLR3G of which their 3'UTRs have similar binding sites for miR-223 with IGF1R 3'UTR. However decoy 1 failed to recover Sp1, CDC27 and FBXW7 expression. These data support that the sequence-specific decoy oligonucleotides might represent exogenous competing RNA which selectively inhibits microRNA targeting.

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Related in: MedlinePlus

Observation of decoy nucleotide influence on miR-223 targeting the 3’UTR by dual luciferase assay.Reporter luciferase activities were measured in the HeLa cells co-transfected with the decoy nucleotides (25 μM), pLL3.7-miR-223, and 3’UTR constructs {psiCHEK-2-3’UTR of IGF1R (A), POLE3G (B), FOXO3 (C), FOXO1 (D), CDC27 (E), FBXW7 (F), PAXIP1 (G)} or the control nucleotide + pLL3.7-miR-223 + psiCHEK-2-3’UTR constructs. Renilla luciferase activities are normalized over firefly luciferase activities and are summarized as mean with the standard deviation. Data are representative from 3 independent experiments. Co. control. **, P<0.01.
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pone-0082167-g002: Observation of decoy nucleotide influence on miR-223 targeting the 3’UTR by dual luciferase assay.Reporter luciferase activities were measured in the HeLa cells co-transfected with the decoy nucleotides (25 μM), pLL3.7-miR-223, and 3’UTR constructs {psiCHEK-2-3’UTR of IGF1R (A), POLE3G (B), FOXO3 (C), FOXO1 (D), CDC27 (E), FBXW7 (F), PAXIP1 (G)} or the control nucleotide + pLL3.7-miR-223 + psiCHEK-2-3’UTR constructs. Renilla luciferase activities are normalized over firefly luciferase activities and are summarized as mean with the standard deviation. Data are representative from 3 independent experiments. Co. control. **, P<0.01.

Mentions: In order to validate the effects of the decoys designed in this study, we detected the expression of endogenous miR-223 and miR-124 by RT-PCR in SMMC-7721 cells. After transfection of the decoys, the level of miR-223 expression was down regulated compared to controls, but miR-124 did not (Figure 1B). We then tested the influence of the decoy nucleotides on miR-223 targeting the 3’UTR based on the pairing between miR-223 and its target sequence. The activities of the decoys were monitored by measuring the relative expression levels of reporter luciferase (Renilla) to their control luciferase (firefly) activities. In each case, as compared to the control cells, transfection of miR-223 indeed significantly inhibited the luciferase activities in all groups of the reporters containing 3’UTR of IGF1R, FOXO3, POLR3G, FOXO1, CDC27, and FBXW7 (Figure 2). While, co-transfection of the decoy oligonucleotide with miR-223 construct led to an increase in the luciferase activity in all assays compared to the group with miR-223 alone, suggesting the effective inhibition of miR-223 activity and expression rescuing of the targets. However, the rescuing effect was not always the same among all these 4 decoy nucleotides. The decoy 1, interestingly, inhibited miR-223 targeting IGF1R 3’UTR more efficiently than the decoy 2 in either HeLa cells (Figure 2A) or HEK-203T cells (data not shown), The inhibitory effect of decoy 1 on miR-223 targeting POLR3G and FOXO3 3’UTR was also similar to IGF1R (Figure 2B,C) as they had the same seed complements. The luciferase activity significantly increased after co-transfection of decoy 1 with miR-223 although the luciferase activity was not recovered to the level of control vector group. There was no significant difference of luciferase activity between the groups of decoy 2 + miR-223 and miR-223 in IGF1R, POLR3G and FOXO3 3’UTR reporter assay. However, decoy 2 had stronger inhibitory effect on miR-223 targeting FOXO1 3’UTR (Figure 2D). Similarly, the decoy 2 recovered CDC27 mRNA 3’UTR from the inhibition by miR-223 more significantly than the decoy 1(Figure 2E) as CDC27 shared two similar additional complementary bases with FOXO1 besides the seed complementary sequence. The rescue effect on FBXW7 3’UTR from the inhibition by miR-223 was quite different from the others. Co-transfection with decoy 1 did not significantly elevate the luciferase activity inhibited by miR-223 (Figure 2F), and only showed slight rescuing effect on FBXW7 3’UTR, but decoys 2 and 3 offered significant rescue effect on FBXW7 3’UTR (Figure 2F). Among all the 3’UTRs that we tested in this study, the relative luciferase activities are highest in the group of decoy 4, suggesting that the nucleotide completely complementary to mature miR-223 was the most potent and strongest rescuer of all the 3’UTRs. Furthermore, decoy 3 containing sequence partially complementary to 3’ region of mature miR-223 had also a robust role in recovering the targets from miR-223 repression (Figure 2). All the decoys had no any effect on the activities of the reporter carrying 3’UTR of PAXIP1 mRNA which was not targeted by miR-223 (Figure 2G), suggesting the rescue effect of the decoy was specific.


Decoy oligonucleotide rescues IGF1R expression from MicroRNA-223 suppression.

Wu LH, Cai QQ, Dong YW, Wang R, He BM, Qi B, Xu CJ, Wu XZ - PLoS ONE (2013)

Observation of decoy nucleotide influence on miR-223 targeting the 3’UTR by dual luciferase assay.Reporter luciferase activities were measured in the HeLa cells co-transfected with the decoy nucleotides (25 μM), pLL3.7-miR-223, and 3’UTR constructs {psiCHEK-2-3’UTR of IGF1R (A), POLE3G (B), FOXO3 (C), FOXO1 (D), CDC27 (E), FBXW7 (F), PAXIP1 (G)} or the control nucleotide + pLL3.7-miR-223 + psiCHEK-2-3’UTR constructs. Renilla luciferase activities are normalized over firefly luciferase activities and are summarized as mean with the standard deviation. Data are representative from 3 independent experiments. Co. control. **, P<0.01.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852755&req=5

pone-0082167-g002: Observation of decoy nucleotide influence on miR-223 targeting the 3’UTR by dual luciferase assay.Reporter luciferase activities were measured in the HeLa cells co-transfected with the decoy nucleotides (25 μM), pLL3.7-miR-223, and 3’UTR constructs {psiCHEK-2-3’UTR of IGF1R (A), POLE3G (B), FOXO3 (C), FOXO1 (D), CDC27 (E), FBXW7 (F), PAXIP1 (G)} or the control nucleotide + pLL3.7-miR-223 + psiCHEK-2-3’UTR constructs. Renilla luciferase activities are normalized over firefly luciferase activities and are summarized as mean with the standard deviation. Data are representative from 3 independent experiments. Co. control. **, P<0.01.
Mentions: In order to validate the effects of the decoys designed in this study, we detected the expression of endogenous miR-223 and miR-124 by RT-PCR in SMMC-7721 cells. After transfection of the decoys, the level of miR-223 expression was down regulated compared to controls, but miR-124 did not (Figure 1B). We then tested the influence of the decoy nucleotides on miR-223 targeting the 3’UTR based on the pairing between miR-223 and its target sequence. The activities of the decoys were monitored by measuring the relative expression levels of reporter luciferase (Renilla) to their control luciferase (firefly) activities. In each case, as compared to the control cells, transfection of miR-223 indeed significantly inhibited the luciferase activities in all groups of the reporters containing 3’UTR of IGF1R, FOXO3, POLR3G, FOXO1, CDC27, and FBXW7 (Figure 2). While, co-transfection of the decoy oligonucleotide with miR-223 construct led to an increase in the luciferase activity in all assays compared to the group with miR-223 alone, suggesting the effective inhibition of miR-223 activity and expression rescuing of the targets. However, the rescuing effect was not always the same among all these 4 decoy nucleotides. The decoy 1, interestingly, inhibited miR-223 targeting IGF1R 3’UTR more efficiently than the decoy 2 in either HeLa cells (Figure 2A) or HEK-203T cells (data not shown), The inhibitory effect of decoy 1 on miR-223 targeting POLR3G and FOXO3 3’UTR was also similar to IGF1R (Figure 2B,C) as they had the same seed complements. The luciferase activity significantly increased after co-transfection of decoy 1 with miR-223 although the luciferase activity was not recovered to the level of control vector group. There was no significant difference of luciferase activity between the groups of decoy 2 + miR-223 and miR-223 in IGF1R, POLR3G and FOXO3 3’UTR reporter assay. However, decoy 2 had stronger inhibitory effect on miR-223 targeting FOXO1 3’UTR (Figure 2D). Similarly, the decoy 2 recovered CDC27 mRNA 3’UTR from the inhibition by miR-223 more significantly than the decoy 1(Figure 2E) as CDC27 shared two similar additional complementary bases with FOXO1 besides the seed complementary sequence. The rescue effect on FBXW7 3’UTR from the inhibition by miR-223 was quite different from the others. Co-transfection with decoy 1 did not significantly elevate the luciferase activity inhibited by miR-223 (Figure 2F), and only showed slight rescuing effect on FBXW7 3’UTR, but decoys 2 and 3 offered significant rescue effect on FBXW7 3’UTR (Figure 2F). Among all the 3’UTRs that we tested in this study, the relative luciferase activities are highest in the group of decoy 4, suggesting that the nucleotide completely complementary to mature miR-223 was the most potent and strongest rescuer of all the 3’UTRs. Furthermore, decoy 3 containing sequence partially complementary to 3’ region of mature miR-223 had also a robust role in recovering the targets from miR-223 repression (Figure 2). All the decoys had no any effect on the activities of the reporter carrying 3’UTR of PAXIP1 mRNA which was not targeted by miR-223 (Figure 2G), suggesting the rescue effect of the decoy was specific.

Bottom Line: All decoys had no effect on PAXIP1 which was not targeted by miR-223.The decoy 1 that was based on the sequence of IGF1R 3'UTR rescued the expression of IGF1R more significantly than other decoy nucleotides except the antisense decoy 4.However decoy 1 failed to recover Sp1, CDC27 and FBXW7 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Children's Health Care, Yu Ying Children's Hospital, Wenzhou Medical College, Wenzhou, China.

ABSTRACT
A mature miRNA generally suppresses hundreds of mRNA targets. To evaluate the selective effect of synthetic oligonucleotide decoys on hsa-miR-223 activity, reporters containing 3' untranslated regions (UTR) of IGF1R, FOXO1, POLR3G, FOXO3, CDC27, FBXW7 and PAXIP1 mRNAs were constructed for the luciferase assay. The oligonucleotide decoys were designed and synthesized according to mature miR-223 sequence and its target mRNA sequence. Quantitative RT-PCR & western analysis were used to measure miR-223-targeted mRNA expression, Interestingly, apart from the antisense oligonucleotide, decoy nucleotides which were complementary to the 5', central or 3' region of mature miR-223 suppressed miR-223 targeting the 3'UTR of IGF1R, FOXO1, FOXO3, CDC27, POLR3G, and FBXW7 mRNAs and rescued the expression of these genes to varying degrees from miR-223 suppression at both mRNA and protein levels. All decoys had no effect on PAXIP1 which was not targeted by miR-223. The decoy 1 that was based on the sequence of IGF1R 3'UTR rescued the expression of IGF1R more significantly than other decoy nucleotides except the antisense decoy 4. Decoy 1 also rescued the expression of FOXO3 and POLR3G of which their 3'UTRs have similar binding sites for miR-223 with IGF1R 3'UTR. However decoy 1 failed to recover Sp1, CDC27 and FBXW7 expression. These data support that the sequence-specific decoy oligonucleotides might represent exogenous competing RNA which selectively inhibits microRNA targeting.

Show MeSH
Related in: MedlinePlus