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A novel dual-fluorescence strategy for functionally validating microRNA targets in 3' untranslated regions: regulation of the inward rectifier potassium channel K(ir)2.1 by miR-212.

Goldoni D, Yarham JM, McGahon MK, O'Connor A, Guduric-Fuchs J, Edgar K, McDonald DM, Simpson DA, Collins A - Biochem. J. (2012)

Bottom Line: The principle of the assay is that functional targeting of the 3'UTR by the microRNA results in a decrease in the red/green fluorescence intensity ratio as determined by automated image analysis.The red/green ratio was lower in miR-212-expressing cells compared with the non-targeting controls, an effect that was attenuated by mutating the predicted target site. miR-212 also reduced inward rectifier current and K(ir)2.1 protein in HeLa cells.This novel assay has several advantages over traditional luciferase-based assays including larger sample size, amenability to time course studies and adaptability to high-throughput screening.

View Article: PubMed Central - PubMed

Affiliation: Centre for Vision and Vascular Science, Queen's University of Belfast, Institute of Clinical Science, Block A, Royal Victoria Hospital, Grosvenor Road, Belfast, BT12 6BA, UK.

ABSTRACT
Gene targeting by microRNAs is important in health and disease. We developed a functional assay for identifying microRNA targets and applied it to the K(+) channel K(ir)2.1 [KCNJ2 (potassium inwardly-rectifying channel, subfamily J, member 2)] which is dysregulated in cardiac and vascular disorders. The 3'UTR (untranslated region) was inserted downstream of the mCherry red fluorescent protein coding sequence in a mammalian expression plasmid. MicroRNA sequences were inserted into the pSM30 expression vector which provides enhanced green fluorescent protein as an indicator of microRNA expression. HEK (human embryonic kidney)-293 cells were co-transfected with the mCherry-3'UTR plasmid and a pSM30-based plasmid with a microRNA insert. The principle of the assay is that functional targeting of the 3'UTR by the microRNA results in a decrease in the red/green fluorescence intensity ratio as determined by automated image analysis. The method was validated with miR-1, a known down-regulator of K(ir)2.1 expression, and was used to investigate the targeting of the K(ir)2.1 3'UTR by miR-212. The red/green ratio was lower in miR-212-expressing cells compared with the non-targeting controls, an effect that was attenuated by mutating the predicted target site. miR-212 also reduced inward rectifier current and K(ir)2.1 protein in HeLa cells. This novel assay has several advantages over traditional luciferase-based assays including larger sample size, amenability to time course studies and adaptability to high-throughput screening.

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Assessment of miR-212 targeting by luciferase assayHEK-293 cells were transfected with pmirGLOKir2.1UTR and pSM30-SCR or pSM30-miR-212. Firefly and Renilla luciferase activities were determined using the Dual-Glo® Luciferase assay. Data are expressed as firefly luciferase activity normalized to Renilla luciferase activity (means±S.E.M.). ***P<.001 as determined by Student's t test on log-transformed data; n=6.
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Figure 5: Assessment of miR-212 targeting by luciferase assayHEK-293 cells were transfected with pmirGLOKir2.1UTR and pSM30-SCR or pSM30-miR-212. Firefly and Renilla luciferase activities were determined using the Dual-Glo® Luciferase assay. Data are expressed as firefly luciferase activity normalized to Renilla luciferase activity (means±S.E.M.). ***P<.001 as determined by Student's t test on log-transformed data; n=6.

Mentions: The novel miR-212 result from the dual-fluorescence assay suggested that a similar result may be obtained with a more traditional luciferase-based assay. The effect of miR-212 on the expression of firefly luciferase with the Kir2.1 3′UTR is shown in Figure 5. Firefly luciferase activity (normalized to Renilla luciferase) was less for pSM30-miR-212- than pSM30-SCR-transfected cells.


A novel dual-fluorescence strategy for functionally validating microRNA targets in 3' untranslated regions: regulation of the inward rectifier potassium channel K(ir)2.1 by miR-212.

Goldoni D, Yarham JM, McGahon MK, O'Connor A, Guduric-Fuchs J, Edgar K, McDonald DM, Simpson DA, Collins A - Biochem. J. (2012)

Assessment of miR-212 targeting by luciferase assayHEK-293 cells were transfected with pmirGLOKir2.1UTR and pSM30-SCR or pSM30-miR-212. Firefly and Renilla luciferase activities were determined using the Dual-Glo® Luciferase assay. Data are expressed as firefly luciferase activity normalized to Renilla luciferase activity (means±S.E.M.). ***P<.001 as determined by Student's t test on log-transformed data; n=6.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475433&req=5

Figure 5: Assessment of miR-212 targeting by luciferase assayHEK-293 cells were transfected with pmirGLOKir2.1UTR and pSM30-SCR or pSM30-miR-212. Firefly and Renilla luciferase activities were determined using the Dual-Glo® Luciferase assay. Data are expressed as firefly luciferase activity normalized to Renilla luciferase activity (means±S.E.M.). ***P<.001 as determined by Student's t test on log-transformed data; n=6.
Mentions: The novel miR-212 result from the dual-fluorescence assay suggested that a similar result may be obtained with a more traditional luciferase-based assay. The effect of miR-212 on the expression of firefly luciferase with the Kir2.1 3′UTR is shown in Figure 5. Firefly luciferase activity (normalized to Renilla luciferase) was less for pSM30-miR-212- than pSM30-SCR-transfected cells.

Bottom Line: The principle of the assay is that functional targeting of the 3'UTR by the microRNA results in a decrease in the red/green fluorescence intensity ratio as determined by automated image analysis.The red/green ratio was lower in miR-212-expressing cells compared with the non-targeting controls, an effect that was attenuated by mutating the predicted target site. miR-212 also reduced inward rectifier current and K(ir)2.1 protein in HeLa cells.This novel assay has several advantages over traditional luciferase-based assays including larger sample size, amenability to time course studies and adaptability to high-throughput screening.

View Article: PubMed Central - PubMed

Affiliation: Centre for Vision and Vascular Science, Queen's University of Belfast, Institute of Clinical Science, Block A, Royal Victoria Hospital, Grosvenor Road, Belfast, BT12 6BA, UK.

ABSTRACT
Gene targeting by microRNAs is important in health and disease. We developed a functional assay for identifying microRNA targets and applied it to the K(+) channel K(ir)2.1 [KCNJ2 (potassium inwardly-rectifying channel, subfamily J, member 2)] which is dysregulated in cardiac and vascular disorders. The 3'UTR (untranslated region) was inserted downstream of the mCherry red fluorescent protein coding sequence in a mammalian expression plasmid. MicroRNA sequences were inserted into the pSM30 expression vector which provides enhanced green fluorescent protein as an indicator of microRNA expression. HEK (human embryonic kidney)-293 cells were co-transfected with the mCherry-3'UTR plasmid and a pSM30-based plasmid with a microRNA insert. The principle of the assay is that functional targeting of the 3'UTR by the microRNA results in a decrease in the red/green fluorescence intensity ratio as determined by automated image analysis. The method was validated with miR-1, a known down-regulator of K(ir)2.1 expression, and was used to investigate the targeting of the K(ir)2.1 3'UTR by miR-212. The red/green ratio was lower in miR-212-expressing cells compared with the non-targeting controls, an effect that was attenuated by mutating the predicted target site. miR-212 also reduced inward rectifier current and K(ir)2.1 protein in HeLa cells. This novel assay has several advantages over traditional luciferase-based assays including larger sample size, amenability to time course studies and adaptability to high-throughput screening.

Show MeSH
Related in: MedlinePlus