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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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Related in: MedlinePlus

Endogenous expression of Kir2.1 in HeLa cellsRT–PCR was performed on RNA extracted from HeLa cells (lanes 5–7). Negative controls were performed without reverse transcription (lanes 8 and 9; −RT). PCR was performed on human genomic DNA (lanes 2–4; gDNA). Primers were specific for human Kir2.1 (lanes 2, 5 and 8), human Kir2.2 (lanes 3, 6 and 9) and 18S ribosomal RNA (lanes 4 and 7). Reaction products were analysed by agarose ethidium bromide gel electrophoresis.
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Figure 6: Endogenous expression of Kir2.1 in HeLa cellsRT–PCR was performed on RNA extracted from HeLa cells (lanes 5–7). Negative controls were performed without reverse transcription (lanes 8 and 9; −RT). PCR was performed on human genomic DNA (lanes 2–4; gDNA). Primers were specific for human Kir2.1 (lanes 2, 5 and 8), human Kir2.2 (lanes 3, 6 and 9) and 18S ribosomal RNA (lanes 4 and 7). Reaction products were analysed by agarose ethidium bromide gel electrophoresis.

Mentions: The results of the dual-fluorescence assay predicted that miR-212 would suppress the expression of endogenous Kir2.1. We first confirmed the expression of Kir2.1 mRNA in HeLa cells, as shown by the results of RT–PCR experiments shown in Figure 6. Primers were designed to amplify Kir2.1, Kir2.2 and 18S ribosomal RNA. Human genomic DNA was used as a positive control for these primers (lanes 2–4). PCR products from HeLa cDNA are shown in lanes 5–7. There was clearly a product for Kir2.1 (lane 5) and a very faint product for Kir2.2 (lane 6) despite a strong signal with the Kir2.2 primers from the genomic DNA template (lane 3).


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)

Endogenous expression of Kir2.1 in HeLa cellsRT–PCR was performed on RNA extracted from HeLa cells (lanes 5–7). Negative controls were performed without reverse transcription (lanes 8 and 9; −RT). PCR was performed on human genomic DNA (lanes 2–4; gDNA). Primers were specific for human Kir2.1 (lanes 2, 5 and 8), human Kir2.2 (lanes 3, 6 and 9) and 18S ribosomal RNA (lanes 4 and 7). Reaction products were analysed by agarose ethidium bromide gel electrophoresis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Endogenous expression of Kir2.1 in HeLa cellsRT–PCR was performed on RNA extracted from HeLa cells (lanes 5–7). Negative controls were performed without reverse transcription (lanes 8 and 9; −RT). PCR was performed on human genomic DNA (lanes 2–4; gDNA). Primers were specific for human Kir2.1 (lanes 2, 5 and 8), human Kir2.2 (lanes 3, 6 and 9) and 18S ribosomal RNA (lanes 4 and 7). Reaction products were analysed by agarose ethidium bromide gel electrophoresis.
Mentions: The results of the dual-fluorescence assay predicted that miR-212 would suppress the expression of endogenous Kir2.1. We first confirmed the expression of Kir2.1 mRNA in HeLa cells, as shown by the results of RT–PCR experiments shown in Figure 6. Primers were designed to amplify Kir2.1, Kir2.2 and 18S ribosomal RNA. Human genomic DNA was used as a positive control for these primers (lanes 2–4). PCR products from HeLa cDNA are shown in lanes 5–7. There was clearly a product for Kir2.1 (lane 5) and a very faint product for Kir2.2 (lane 6) despite a strong signal with the Kir2.2 primers from the genomic DNA template (lane 3).

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