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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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Inward-rectifier K+ currents in HeLa cells transfected with pSM30-miR-212, pSM30-KCNJ2si or pSM30-SCR(A) The voltage protocol used to elicit currents. (B) Whole-cell currents elicited in the absence (ctrl) and presence of 100 μM Ba2+ (Ba2+) in cells 48 h after transfection with pSM30-SCR (i) and pSM30-miR-212 (ii). (C) Mean±S.E.M. whole-cell current-densities (normalized to cell capacitance and corrected for junction potential) recorded in 8 cells transfected with pSM30-SCR (i) and 13 cells transfected with pSM30-miR-212(ii). (D) Mean±S.E.M. Ba2+-sensitive current densities for pSM30-SCR- and pSM30-miR-212-transfected cells. (E) Mean±S.E.M. Ba2+-sensitive current densities for pSM30-SCR- (n=11) and pSM30-KCNJ2si-transfected (n=10) cells.*P<0.05, **P<0.01 and ***P<0.001 as determined by ANOVA with a Bonferroni-corrected ad hoc test.
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Figure 9: Inward-rectifier K+ currents in HeLa cells transfected with pSM30-miR-212, pSM30-KCNJ2si or pSM30-SCR(A) The voltage protocol used to elicit currents. (B) Whole-cell currents elicited in the absence (ctrl) and presence of 100 μM Ba2+ (Ba2+) in cells 48 h after transfection with pSM30-SCR (i) and pSM30-miR-212 (ii). (C) Mean±S.E.M. whole-cell current-densities (normalized to cell capacitance and corrected for junction potential) recorded in 8 cells transfected with pSM30-SCR (i) and 13 cells transfected with pSM30-miR-212(ii). (D) Mean±S.E.M. Ba2+-sensitive current densities for pSM30-SCR- and pSM30-miR-212-transfected cells. (E) Mean±S.E.M. Ba2+-sensitive current densities for pSM30-SCR- (n=11) and pSM30-KCNJ2si-transfected (n=10) cells.*P<0.05, **P<0.01 and ***P<0.001 as determined by ANOVA with a Bonferroni-corrected ad hoc test.

Mentions: We sought further evidence for targeting of Kir2.1 by miR-212 by investigating the effect on inward-rectifier K+ channel activity itself in HeLa cells. Green fluorescent HeLa cells were selected 48 h post-transfection with pSM30-miR-212 or pSM30-SCR for measurement of inwardly-rectifying K+ current using the patch-clamp technique. Voltage ramps (−100 mV to +50 mV at 60 mV/s; Figure 9A) applied from a holding potential of −40 mV resulted in a current which was inward at negative voltages and reversed close to 0 mV (see Figure 9Bi; ctrl). Application of 100 μM Ba2+ (a relatively selective inhibitor of inward rectifier K+ channels [30–33]) inhibited portions of both the inward and outward whole-cell current with preferential inhibition of the inward portion (Figure 9Bi; Ba2+). On average the current at −108 mV (corrected for pipette junction potential) recorded in pSM30-SCR transfected cells was reduced from −219.02±47.61 pA/pF to −178.71±47.79 pA/pF by the application of 100 μM Ba2+ (Figure 9Ci; n=8; P<0.01 determined by paired Student's t test). As plotted in Figure 9(D) the Ba2+-sensitive current exhibited strong inward rectification and reversed at −3.55±2.61 mV. In cells transfected with pSM30-miR-212 the whole-cell inward current was smaller in amplitude to that in cells transfected with pSM30-SCR (Figure 9Bii). On average the current at −108 mV (corrected for pipette junction potential) recorded in pSM30-miR-212-transfected cells was reduced from −104.98±41.07 pA/pF to −100.91±41.10 pA/pF by the application of 100 μM Ba2+ (Figure 9Cii; n=13; P<0.01 determined by paired Student's t test). The Ba2+-sensitive current in cells transfected with pSM30-miR-212 was significantly smaller than the equivalent current in pSM30-SCR-transfected cells (Figure 9D; P<0.01 determined by ANOVA for the entire IV relationship). Transfection with pSM30-KCNJ2si also reduced the Ba2+-sensitive current [Figure 9E; P<0.01 determined by ANOVA for the entire IV (current–voltage) relationship].


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)

Inward-rectifier K+ currents in HeLa cells transfected with pSM30-miR-212, pSM30-KCNJ2si or pSM30-SCR(A) The voltage protocol used to elicit currents. (B) Whole-cell currents elicited in the absence (ctrl) and presence of 100 μM Ba2+ (Ba2+) in cells 48 h after transfection with pSM30-SCR (i) and pSM30-miR-212 (ii). (C) Mean±S.E.M. whole-cell current-densities (normalized to cell capacitance and corrected for junction potential) recorded in 8 cells transfected with pSM30-SCR (i) and 13 cells transfected with pSM30-miR-212(ii). (D) Mean±S.E.M. Ba2+-sensitive current densities for pSM30-SCR- and pSM30-miR-212-transfected cells. (E) Mean±S.E.M. Ba2+-sensitive current densities for pSM30-SCR- (n=11) and pSM30-KCNJ2si-transfected (n=10) cells.*P<0.05, **P<0.01 and ***P<0.001 as determined by ANOVA with a Bonferroni-corrected ad hoc test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Inward-rectifier K+ currents in HeLa cells transfected with pSM30-miR-212, pSM30-KCNJ2si or pSM30-SCR(A) The voltage protocol used to elicit currents. (B) Whole-cell currents elicited in the absence (ctrl) and presence of 100 μM Ba2+ (Ba2+) in cells 48 h after transfection with pSM30-SCR (i) and pSM30-miR-212 (ii). (C) Mean±S.E.M. whole-cell current-densities (normalized to cell capacitance and corrected for junction potential) recorded in 8 cells transfected with pSM30-SCR (i) and 13 cells transfected with pSM30-miR-212(ii). (D) Mean±S.E.M. Ba2+-sensitive current densities for pSM30-SCR- and pSM30-miR-212-transfected cells. (E) Mean±S.E.M. Ba2+-sensitive current densities for pSM30-SCR- (n=11) and pSM30-KCNJ2si-transfected (n=10) cells.*P<0.05, **P<0.01 and ***P<0.001 as determined by ANOVA with a Bonferroni-corrected ad hoc test.
Mentions: We sought further evidence for targeting of Kir2.1 by miR-212 by investigating the effect on inward-rectifier K+ channel activity itself in HeLa cells. Green fluorescent HeLa cells were selected 48 h post-transfection with pSM30-miR-212 or pSM30-SCR for measurement of inwardly-rectifying K+ current using the patch-clamp technique. Voltage ramps (−100 mV to +50 mV at 60 mV/s; Figure 9A) applied from a holding potential of −40 mV resulted in a current which was inward at negative voltages and reversed close to 0 mV (see Figure 9Bi; ctrl). Application of 100 μM Ba2+ (a relatively selective inhibitor of inward rectifier K+ channels [30–33]) inhibited portions of both the inward and outward whole-cell current with preferential inhibition of the inward portion (Figure 9Bi; Ba2+). On average the current at −108 mV (corrected for pipette junction potential) recorded in pSM30-SCR transfected cells was reduced from −219.02±47.61 pA/pF to −178.71±47.79 pA/pF by the application of 100 μM Ba2+ (Figure 9Ci; n=8; P<0.01 determined by paired Student's t test). As plotted in Figure 9(D) the Ba2+-sensitive current exhibited strong inward rectification and reversed at −3.55±2.61 mV. In cells transfected with pSM30-miR-212 the whole-cell inward current was smaller in amplitude to that in cells transfected with pSM30-SCR (Figure 9Bii). On average the current at −108 mV (corrected for pipette junction potential) recorded in pSM30-miR-212-transfected cells was reduced from −104.98±41.07 pA/pF to −100.91±41.10 pA/pF by the application of 100 μM Ba2+ (Figure 9Cii; n=13; P<0.01 determined by paired Student's t test). The Ba2+-sensitive current in cells transfected with pSM30-miR-212 was significantly smaller than the equivalent current in pSM30-SCR-transfected cells (Figure 9D; P<0.01 determined by ANOVA for the entire IV relationship). Transfection with pSM30-KCNJ2si also reduced the Ba2+-sensitive current [Figure 9E; P<0.01 determined by ANOVA for the entire IV (current–voltage) relationship].

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