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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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Results of image analysis two days after co-transfection of HEK-293 cellsDown-regulation of mCherry-Kir2.1 3′UTR by pSM30-miR-1 and pSM30-miR-212, and attenuation of the miR-212 effect by mutation of the putative seed region. (A) Green compared with red integrated fluorescence intensity of cells transfected with pSM30-SCR (○) and pSM30-miR-1 (□). Lines are linear regressions. (B) Relative frequency histograms of log red/green intensity ratios derived from the data in (A). Lines are fits of Gaussian distributions. (C) Green compared with red integrated fluorescence intensity of cells transfected with pSM30-SCR (○) and pSM30-miR-212 (□). Results are from the same experiment as (A) and are presented in a separate panel for ease of viewing; pSM30-SCR data are the same in (A) and (C). Lines are linear regressions. (D) Red/green intensity ratios (means±S.E.M.) for pSM30-SCR-, pSM30-miR-1- and pSM30-miR-212-transfected cells. Log-transformed data were compared by one-way ANOVA; ***P<0.001 for miR-1 compared with SCR and miR-212 compared with SCR; n=731 (SCR), 776 (miR-1) and 550 (miR-212). (E) Red/green ratio in pSM30-miR-212-transfected cells as a percentage of red/green ratio in pSM30-SCR-transfected cells (means±S.E.M.). Co-transfections were with pmChKir2.1UTR (Wild-type) or pmChKir2.1UTRm212 (Mutant). *P<.05 as determined by Student's t test on the log-transformed percentage decrease for 3 independent experiments.
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Figure 3: Results of image analysis two days after co-transfection of HEK-293 cellsDown-regulation of mCherry-Kir2.1 3′UTR by pSM30-miR-1 and pSM30-miR-212, and attenuation of the miR-212 effect by mutation of the putative seed region. (A) Green compared with red integrated fluorescence intensity of cells transfected with pSM30-SCR (○) and pSM30-miR-1 (□). Lines are linear regressions. (B) Relative frequency histograms of log red/green intensity ratios derived from the data in (A). Lines are fits of Gaussian distributions. (C) Green compared with red integrated fluorescence intensity of cells transfected with pSM30-SCR (○) and pSM30-miR-212 (□). Results are from the same experiment as (A) and are presented in a separate panel for ease of viewing; pSM30-SCR data are the same in (A) and (C). Lines are linear regressions. (D) Red/green intensity ratios (means±S.E.M.) for pSM30-SCR-, pSM30-miR-1- and pSM30-miR-212-transfected cells. Log-transformed data were compared by one-way ANOVA; ***P<0.001 for miR-1 compared with SCR and miR-212 compared with SCR; n=731 (SCR), 776 (miR-1) and 550 (miR-212). (E) Red/green ratio in pSM30-miR-212-transfected cells as a percentage of red/green ratio in pSM30-SCR-transfected cells (means±S.E.M.). Co-transfections were with pmChKir2.1UTR (Wild-type) or pmChKir2.1UTRm212 (Mutant). *P<.05 as determined by Student's t test on the log-transformed percentage decrease for 3 independent experiments.

Mentions: If a microRNA co-expressed with EGFP targets the 3′UTR cloned downstream of the red fluorescent protein coding sequence (mCherry) we would expect to observe a decrease in red fluorescence and a concomitant reduction in the ratio of red/green fluorescence. HEK-293 cells were therefore co-transfected with pmChKir2.1UTR and a pSM30-based plasmid expressing either miR-1 or a non-targeting miRNA control (SCR). The total green and red intensities of multiple cells were measured using fluorescent microscopy and the values for each cell plotted against each other (Figure 3A). Comparison of the lines of best fit clearly illustrates the expected reduction in red fluorescence in those cells overexpressing miR-1 compared with the non-targeting miRNA. Similarly, the relative frequency histograms of log red/green fluorescence ratios for cells transfected with pmChKir2.1UTR/pSM30-miR-1 are shifted to the left-hand side relative to those for cells transfected with pmChKir2.1UTR/pSM30-SCR (Figure 3B). A plot of red compared with green intensity for cells transfected with pmChKir2.1UTR/pSM30-miR-212 and pmChKir2.1UTR/pSM30-SCR is shown in Figure 3(C). Red/green fluorescence was significantly lower for pmChKir2.1UTR/pSM30-miR-1-transfected and pmChKir2.1UTR/pSM30-miR-212-transfected compared with the pmChKir2.1UTR/pSM30-SCR-transfected cells (Figure 3D).


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)

Results of image analysis two days after co-transfection of HEK-293 cellsDown-regulation of mCherry-Kir2.1 3′UTR by pSM30-miR-1 and pSM30-miR-212, and attenuation of the miR-212 effect by mutation of the putative seed region. (A) Green compared with red integrated fluorescence intensity of cells transfected with pSM30-SCR (○) and pSM30-miR-1 (□). Lines are linear regressions. (B) Relative frequency histograms of log red/green intensity ratios derived from the data in (A). Lines are fits of Gaussian distributions. (C) Green compared with red integrated fluorescence intensity of cells transfected with pSM30-SCR (○) and pSM30-miR-212 (□). Results are from the same experiment as (A) and are presented in a separate panel for ease of viewing; pSM30-SCR data are the same in (A) and (C). Lines are linear regressions. (D) Red/green intensity ratios (means±S.E.M.) for pSM30-SCR-, pSM30-miR-1- and pSM30-miR-212-transfected cells. Log-transformed data were compared by one-way ANOVA; ***P<0.001 for miR-1 compared with SCR and miR-212 compared with SCR; n=731 (SCR), 776 (miR-1) and 550 (miR-212). (E) Red/green ratio in pSM30-miR-212-transfected cells as a percentage of red/green ratio in pSM30-SCR-transfected cells (means±S.E.M.). Co-transfections were with pmChKir2.1UTR (Wild-type) or pmChKir2.1UTRm212 (Mutant). *P<.05 as determined by Student's t test on the log-transformed percentage decrease for 3 independent experiments.
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Figure 3: Results of image analysis two days after co-transfection of HEK-293 cellsDown-regulation of mCherry-Kir2.1 3′UTR by pSM30-miR-1 and pSM30-miR-212, and attenuation of the miR-212 effect by mutation of the putative seed region. (A) Green compared with red integrated fluorescence intensity of cells transfected with pSM30-SCR (○) and pSM30-miR-1 (□). Lines are linear regressions. (B) Relative frequency histograms of log red/green intensity ratios derived from the data in (A). Lines are fits of Gaussian distributions. (C) Green compared with red integrated fluorescence intensity of cells transfected with pSM30-SCR (○) and pSM30-miR-212 (□). Results are from the same experiment as (A) and are presented in a separate panel for ease of viewing; pSM30-SCR data are the same in (A) and (C). Lines are linear regressions. (D) Red/green intensity ratios (means±S.E.M.) for pSM30-SCR-, pSM30-miR-1- and pSM30-miR-212-transfected cells. Log-transformed data were compared by one-way ANOVA; ***P<0.001 for miR-1 compared with SCR and miR-212 compared with SCR; n=731 (SCR), 776 (miR-1) and 550 (miR-212). (E) Red/green ratio in pSM30-miR-212-transfected cells as a percentage of red/green ratio in pSM30-SCR-transfected cells (means±S.E.M.). Co-transfections were with pmChKir2.1UTR (Wild-type) or pmChKir2.1UTRm212 (Mutant). *P<.05 as determined by Student's t test on the log-transformed percentage decrease for 3 independent experiments.
Mentions: If a microRNA co-expressed with EGFP targets the 3′UTR cloned downstream of the red fluorescent protein coding sequence (mCherry) we would expect to observe a decrease in red fluorescence and a concomitant reduction in the ratio of red/green fluorescence. HEK-293 cells were therefore co-transfected with pmChKir2.1UTR and a pSM30-based plasmid expressing either miR-1 or a non-targeting miRNA control (SCR). The total green and red intensities of multiple cells were measured using fluorescent microscopy and the values for each cell plotted against each other (Figure 3A). Comparison of the lines of best fit clearly illustrates the expected reduction in red fluorescence in those cells overexpressing miR-1 compared with the non-targeting miRNA. Similarly, the relative frequency histograms of log red/green fluorescence ratios for cells transfected with pmChKir2.1UTR/pSM30-miR-1 are shifted to the left-hand side relative to those for cells transfected with pmChKir2.1UTR/pSM30-SCR (Figure 3B). A plot of red compared with green intensity for cells transfected with pmChKir2.1UTR/pSM30-miR-212 and pmChKir2.1UTR/pSM30-SCR is shown in Figure 3(C). Red/green fluorescence was significantly lower for pmChKir2.1UTR/pSM30-miR-1-transfected and pmChKir2.1UTR/pSM30-miR-212-transfected compared with the pmChKir2.1UTR/pSM30-SCR-transfected cells (Figure 3D).

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