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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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Characterization of stably transfected mChKir2.1UTR cells and down-regulation of mCherry-Kir2.1 3′UTR expression by miR-212(A) Confirmation of integration of the mCherry-Kir2.1 3′UTR construct in the genome of mChKir2.1UTR cells. Agarose ethidium bromide electrophoresis of PCRs using primers described in the Experimental section and templates as follows: lane 2: mChKir2.1UTR genomic DNA; lane 3: HEK293 genomic DNA; lane 4: no-template control; lane 5: pmChKir2.1UTR plasmid. (B) Down-regulation of mCherry expression in mChKir2.1UTR cells by miR-212. Red/green intensity ratios (means±S.E.M.) for pSM30-SCR-transfected (SCR) and pSM30-miR-212-transfected (miR-212) cells. Log-transformed data were compared by Student's t test; ***P<0.001; n=801 (SCR) and 478 (miR-212).
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Figure 4: Characterization of stably transfected mChKir2.1UTR cells and down-regulation of mCherry-Kir2.1 3′UTR expression by miR-212(A) Confirmation of integration of the mCherry-Kir2.1 3′UTR construct in the genome of mChKir2.1UTR cells. Agarose ethidium bromide electrophoresis of PCRs using primers described in the Experimental section and templates as follows: lane 2: mChKir2.1UTR genomic DNA; lane 3: HEK293 genomic DNA; lane 4: no-template control; lane 5: pmChKir2.1UTR plasmid. (B) Down-regulation of mCherry expression in mChKir2.1UTR cells by miR-212. Red/green intensity ratios (means±S.E.M.) for pSM30-SCR-transfected (SCR) and pSM30-miR-212-transfected (miR-212) cells. Log-transformed data were compared by Student's t test; ***P<0.001; n=801 (SCR) and 478 (miR-212).

Mentions: The use of a reporter plasmid in addition to the miRNA expression vector has some potential drawbacks. For example variations in transfection efficiency may introduce noise that multiplies with the number of co-transfected constructs. A strategy that employs single transfections may abrogate this problem. We therefore generated a cell line stably transfected with the mCherry reporter linked with the Kir2.1 3′UTR (mChKir2.1UTR cells). This was confirmed by PCR analysis of genomic DNA. Figure 4(A) shows an agarose ethidium bromide electrophoresis gel of PCR products from mChKir2.1UTR genomic DNA (lane 2), untransfected HEK-293 genomic DNA (lane 3), no-template control (lane 4) and pmChKir2.1UTR plasmid (lane 5). The forward primer site is in the mCherry coding sequence and the reverse primer site is at the end of the Kir2.1 3′UTR sequence (Figure 1A). The bands in lanes 2 and 5 are consistent with the predicted product size of 4055 bp. These data confirm the presence of the Kir2.1 3′UTR downstream of the mCherry coding sequence in the genomic DNA of mChKir2.1UTR 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)

Characterization of stably transfected mChKir2.1UTR cells and down-regulation of mCherry-Kir2.1 3′UTR expression by miR-212(A) Confirmation of integration of the mCherry-Kir2.1 3′UTR construct in the genome of mChKir2.1UTR cells. Agarose ethidium bromide electrophoresis of PCRs using primers described in the Experimental section and templates as follows: lane 2: mChKir2.1UTR genomic DNA; lane 3: HEK293 genomic DNA; lane 4: no-template control; lane 5: pmChKir2.1UTR plasmid. (B) Down-regulation of mCherry expression in mChKir2.1UTR cells by miR-212. Red/green intensity ratios (means±S.E.M.) for pSM30-SCR-transfected (SCR) and pSM30-miR-212-transfected (miR-212) cells. Log-transformed data were compared by Student's t test; ***P<0.001; n=801 (SCR) and 478 (miR-212).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Characterization of stably transfected mChKir2.1UTR cells and down-regulation of mCherry-Kir2.1 3′UTR expression by miR-212(A) Confirmation of integration of the mCherry-Kir2.1 3′UTR construct in the genome of mChKir2.1UTR cells. Agarose ethidium bromide electrophoresis of PCRs using primers described in the Experimental section and templates as follows: lane 2: mChKir2.1UTR genomic DNA; lane 3: HEK293 genomic DNA; lane 4: no-template control; lane 5: pmChKir2.1UTR plasmid. (B) Down-regulation of mCherry expression in mChKir2.1UTR cells by miR-212. Red/green intensity ratios (means±S.E.M.) for pSM30-SCR-transfected (SCR) and pSM30-miR-212-transfected (miR-212) cells. Log-transformed data were compared by Student's t test; ***P<0.001; n=801 (SCR) and 478 (miR-212).
Mentions: The use of a reporter plasmid in addition to the miRNA expression vector has some potential drawbacks. For example variations in transfection efficiency may introduce noise that multiplies with the number of co-transfected constructs. A strategy that employs single transfections may abrogate this problem. We therefore generated a cell line stably transfected with the mCherry reporter linked with the Kir2.1 3′UTR (mChKir2.1UTR cells). This was confirmed by PCR analysis of genomic DNA. Figure 4(A) shows an agarose ethidium bromide electrophoresis gel of PCR products from mChKir2.1UTR genomic DNA (lane 2), untransfected HEK-293 genomic DNA (lane 3), no-template control (lane 4) and pmChKir2.1UTR plasmid (lane 5). The forward primer site is in the mCherry coding sequence and the reverse primer site is at the end of the Kir2.1 3′UTR sequence (Figure 1A). The bands in lanes 2 and 5 are consistent with the predicted product size of 4055 bp. These data confirm the presence of the Kir2.1 3′UTR downstream of the mCherry coding sequence in the genomic DNA of mChKir2.1UTR 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