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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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Expression of mature miR-212 in pSM30-miR-212-transfected cellsHEK-293 (A) and HeLa (B) cells were transfected with pSM30-miR-212 (miR-212) or pSM30-SCR (SCR). High-efficiency transfection was confirmed by monitoring green fluorescence. RNA was extracted and qRT–PCR was performed with a miR-212-specific primer as described in Experimental section. A negative control (NTC) was performed without template. The fold difference relative to 5S RNA is expressed as 2−ΔCt (means±S.E.M.). ***P<.001 for miR-212 compared with SCR and NTC as determined by one-way ANOVA on the ΔCt values; n=3.
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Figure 2: Expression of mature miR-212 in pSM30-miR-212-transfected cellsHEK-293 (A) and HeLa (B) cells were transfected with pSM30-miR-212 (miR-212) or pSM30-SCR (SCR). High-efficiency transfection was confirmed by monitoring green fluorescence. RNA was extracted and qRT–PCR was performed with a miR-212-specific primer as described in Experimental section. A negative control (NTC) was performed without template. The fold difference relative to 5S RNA is expressed as 2−ΔCt (means±S.E.M.). ***P<.001 for miR-212 compared with SCR and NTC as determined by one-way ANOVA on the ΔCt values; n=3.

Mentions: The ability of the pSM30 system to direct expression of a mature miRNA was confirmed by qRT–PCR as shown in Figure 2. Transfection with pmiR-212 increased expression of mature miR-212 in HEK-293 (Figure 2A) and in HeLa (Figure 2B) 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)

Expression of mature miR-212 in pSM30-miR-212-transfected cellsHEK-293 (A) and HeLa (B) cells were transfected with pSM30-miR-212 (miR-212) or pSM30-SCR (SCR). High-efficiency transfection was confirmed by monitoring green fluorescence. RNA was extracted and qRT–PCR was performed with a miR-212-specific primer as described in Experimental section. A negative control (NTC) was performed without template. The fold difference relative to 5S RNA is expressed as 2−ΔCt (means±S.E.M.). ***P<.001 for miR-212 compared with SCR and NTC as determined by one-way ANOVA on the ΔCt values; n=3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Expression of mature miR-212 in pSM30-miR-212-transfected cellsHEK-293 (A) and HeLa (B) cells were transfected with pSM30-miR-212 (miR-212) or pSM30-SCR (SCR). High-efficiency transfection was confirmed by monitoring green fluorescence. RNA was extracted and qRT–PCR was performed with a miR-212-specific primer as described in Experimental section. A negative control (NTC) was performed without template. The fold difference relative to 5S RNA is expressed as 2−ΔCt (means±S.E.M.). ***P<.001 for miR-212 compared with SCR and NTC as determined by one-way ANOVA on the ΔCt values; n=3.
Mentions: The ability of the pSM30 system to direct expression of a mature miRNA was confirmed by qRT–PCR as shown in Figure 2. Transfection with pmiR-212 increased expression of mature miR-212 in HEK-293 (Figure 2A) and in HeLa (Figure 2B) 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