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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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No effect of miR-212 on Kir2.1 mRNA in HeLa cellsRNA extraction, reverse transcription and quantitative PCR (TaqMan®) were carried out on non-transfected HeLa cells (NT) and HeLa cells transfected with pSM30-SCR (SCR) or pSM30-miR-212 (miR-212). Data are expressed as 2−ΔCt where ΔCt=Ct(KCNJ2)−Ct(control gene). The control genes were GAPDH (A) and HPRT1 (B). P>0.05 by ANOVA.
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Figure 7: No effect of miR-212 on Kir2.1 mRNA in HeLa cellsRNA extraction, reverse transcription and quantitative PCR (TaqMan®) were carried out on non-transfected HeLa cells (NT) and HeLa cells transfected with pSM30-SCR (SCR) or pSM30-miR-212 (miR-212). Data are expressed as 2−ΔCt where ΔCt=Ct(KCNJ2)−Ct(control gene). The control genes were GAPDH (A) and HPRT1 (B). P>0.05 by ANOVA.

Mentions: MicroRNAs have been reported to promote degradation of the target mRNA in some cases. We performed qPCR to determine whether miR-212 caused a reduction of Kir2.1 mRNA in HeLa cells. As shown in Figure 7, miR-212 expression did not significantly change Kir2.1 mRNA content relative to two different endogenous control genes, GAPDH (Figure 7A) and HPRT1 (Figure 7B). Despite the lack of effect at the mRNA level, miR-212 did down-regulate endogenous Kir2.1 protein in HeLa cells. Figure 8(A) shows an example of a Western blot of membrane extracts from HeLa cells transfected with pSM30-miR-212 or pSM30-SCR, probed with antibodies against Kir2.1 and Na+/K+-ATPase α1 subunit (loading control). Figure 8(B) summarizes the data from three such experiments and shows that miR-212 reduced Kir2.1 protein.


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)

No effect of miR-212 on Kir2.1 mRNA in HeLa cellsRNA extraction, reverse transcription and quantitative PCR (TaqMan®) were carried out on non-transfected HeLa cells (NT) and HeLa cells transfected with pSM30-SCR (SCR) or pSM30-miR-212 (miR-212). Data are expressed as 2−ΔCt where ΔCt=Ct(KCNJ2)−Ct(control gene). The control genes were GAPDH (A) and HPRT1 (B). P>0.05 by ANOVA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: No effect of miR-212 on Kir2.1 mRNA in HeLa cellsRNA extraction, reverse transcription and quantitative PCR (TaqMan®) were carried out on non-transfected HeLa cells (NT) and HeLa cells transfected with pSM30-SCR (SCR) or pSM30-miR-212 (miR-212). Data are expressed as 2−ΔCt where ΔCt=Ct(KCNJ2)−Ct(control gene). The control genes were GAPDH (A) and HPRT1 (B). P>0.05 by ANOVA.
Mentions: MicroRNAs have been reported to promote degradation of the target mRNA in some cases. We performed qPCR to determine whether miR-212 caused a reduction of Kir2.1 mRNA in HeLa cells. As shown in Figure 7, miR-212 expression did not significantly change Kir2.1 mRNA content relative to two different endogenous control genes, GAPDH (Figure 7A) and HPRT1 (Figure 7B). Despite the lack of effect at the mRNA level, miR-212 did down-regulate endogenous Kir2.1 protein in HeLa cells. Figure 8(A) shows an example of a Western blot of membrane extracts from HeLa cells transfected with pSM30-miR-212 or pSM30-SCR, probed with antibodies against Kir2.1 and Na+/K+-ATPase α1 subunit (loading control). Figure 8(B) summarizes the data from three such experiments and shows that miR-212 reduced Kir2.1 protein.

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