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A functional assay for microRNA target identification and validation.

Gäken J, Mohamedali AM, Jiang J, Malik F, Stangl D, Smith AE, Chronis C, Kulasekararaj AG, Thomas NS, Farzaneh F, Tavassoli M, Mufti GJ - Nucleic Acids Res. (2012)

Bottom Line: MicroRNAs (miRNA) are a class of small RNA molecules that regulate numerous critical cellular processes and bind to partially complementary sequences resulting in down-regulation of their target genes.To enable the identification of biologically relevant miRNA targets, we describe a novel functional assay based on a 3'-UTR-enriched library and a positive/negative selection strategy.As proof of principle we have used mir-130a and its validated target MAFB to test this strategy.

View Article: PubMed Central - PubMed

Affiliation: Department of Haematological Medicine, King's College London, Rayne Institute, London SE5 9NU, UK. joop.gaken@kcl.ac.uk

ABSTRACT
MicroRNAs (miRNA) are a class of small RNA molecules that regulate numerous critical cellular processes and bind to partially complementary sequences resulting in down-regulation of their target genes. Due to the incomplete homology of the miRNA to its target site identification of miRNA target genes is difficult and currently based on computational algorithms predicting large numbers of potential targets for a given miRNA. To enable the identification of biologically relevant miRNA targets, we describe a novel functional assay based on a 3'-UTR-enriched library and a positive/negative selection strategy. As proof of principle we have used mir-130a and its validated target MAFB to test this strategy. Identification of MAFB and five additional targets and their subsequent confirmation as mir-130a targets by western blot analysis and knockdown experiments validates this strategy for the functional identification of miRNA targets.

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Inhibition of mir-130a expression by MIRIDIAN hairpin inhibitors. (A) MCF7-mir-130a cells were either mock transfected (mir130aL) or transfected with MIRIDIAN hairpin inhibitors (Dharmacon). Cells were transfected with control hairpin inhibitor (mir130C), or two hairpin inhibitors (mir130a1, mir130a2) directed against mir-130a. After 48 h protein extracts were prepared and western blot analysis with anti-TPT1 was performed. Knockdown of mir-130a restores the TPT1 expression to ∼80–90% of the control MCF7 expression. (B) NIH/3T3 cells, which express high levels of mir-130a, were treated the same as MCF7 cells above. Knockdown of endogenous mir-130a results in a 3- to 4-fold up-regulation of TPT1 protein.
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gks145-F5: Inhibition of mir-130a expression by MIRIDIAN hairpin inhibitors. (A) MCF7-mir-130a cells were either mock transfected (mir130aL) or transfected with MIRIDIAN hairpin inhibitors (Dharmacon). Cells were transfected with control hairpin inhibitor (mir130C), or two hairpin inhibitors (mir130a1, mir130a2) directed against mir-130a. After 48 h protein extracts were prepared and western blot analysis with anti-TPT1 was performed. Knockdown of mir-130a restores the TPT1 expression to ∼80–90% of the control MCF7 expression. (B) NIH/3T3 cells, which express high levels of mir-130a, were treated the same as MCF7 cells above. Knockdown of endogenous mir-130a results in a 3- to 4-fold up-regulation of TPT1 protein.

Mentions: To further investigate TPT1 as a target for mir-130a, MCF7 cells expressing exogenous mir-130a were transfected with two MIRIDIAN hairpin inhibitors directed against mir-130a or a control hairpin inhibitor (Dharmacon). Cells were transfected using RNAiMAX transfection reagent (Invitrogen) and after 48 h analysed for protein expression. Western blot analysis demonstrated a 3- to 4-fold up-regulation of TPT1 expression in MCF7-mir-130a cells when treated with the hairpin inhibitors but not in cells treated with the control hairpin inhibitor or mock transfected cells (Figure 5A). Similarly NIH/3T3 cells, which express high levels of endogenous mir-130a (Supplementary Figure S2), were treated with the MIRIDIAN hairpin inhibitors. Western blot analysis 48 h after transfection showed a 3- to 4-fold up-regulation of TPT1 protein in these cells, validating TPT1 as a target for mir-130a (Figure 5B). Western blot analysis of the same NIH/3T3 protein extracts showed that the inhibition of mir-130a also resulted in the up-regulation of two other identified targets KIFAP3 and CYP27A1, although to a lesser extent, 2- and 0.4-fold, respectively (Supplementary Figure S7).Figure 5.


A functional assay for microRNA target identification and validation.

Gäken J, Mohamedali AM, Jiang J, Malik F, Stangl D, Smith AE, Chronis C, Kulasekararaj AG, Thomas NS, Farzaneh F, Tavassoli M, Mufti GJ - Nucleic Acids Res. (2012)

Inhibition of mir-130a expression by MIRIDIAN hairpin inhibitors. (A) MCF7-mir-130a cells were either mock transfected (mir130aL) or transfected with MIRIDIAN hairpin inhibitors (Dharmacon). Cells were transfected with control hairpin inhibitor (mir130C), or two hairpin inhibitors (mir130a1, mir130a2) directed against mir-130a. After 48 h protein extracts were prepared and western blot analysis with anti-TPT1 was performed. Knockdown of mir-130a restores the TPT1 expression to ∼80–90% of the control MCF7 expression. (B) NIH/3T3 cells, which express high levels of mir-130a, were treated the same as MCF7 cells above. Knockdown of endogenous mir-130a results in a 3- to 4-fold up-regulation of TPT1 protein.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3378903&req=5

gks145-F5: Inhibition of mir-130a expression by MIRIDIAN hairpin inhibitors. (A) MCF7-mir-130a cells were either mock transfected (mir130aL) or transfected with MIRIDIAN hairpin inhibitors (Dharmacon). Cells were transfected with control hairpin inhibitor (mir130C), or two hairpin inhibitors (mir130a1, mir130a2) directed against mir-130a. After 48 h protein extracts were prepared and western blot analysis with anti-TPT1 was performed. Knockdown of mir-130a restores the TPT1 expression to ∼80–90% of the control MCF7 expression. (B) NIH/3T3 cells, which express high levels of mir-130a, were treated the same as MCF7 cells above. Knockdown of endogenous mir-130a results in a 3- to 4-fold up-regulation of TPT1 protein.
Mentions: To further investigate TPT1 as a target for mir-130a, MCF7 cells expressing exogenous mir-130a were transfected with two MIRIDIAN hairpin inhibitors directed against mir-130a or a control hairpin inhibitor (Dharmacon). Cells were transfected using RNAiMAX transfection reagent (Invitrogen) and after 48 h analysed for protein expression. Western blot analysis demonstrated a 3- to 4-fold up-regulation of TPT1 expression in MCF7-mir-130a cells when treated with the hairpin inhibitors but not in cells treated with the control hairpin inhibitor or mock transfected cells (Figure 5A). Similarly NIH/3T3 cells, which express high levels of endogenous mir-130a (Supplementary Figure S2), were treated with the MIRIDIAN hairpin inhibitors. Western blot analysis 48 h after transfection showed a 3- to 4-fold up-regulation of TPT1 protein in these cells, validating TPT1 as a target for mir-130a (Figure 5B). Western blot analysis of the same NIH/3T3 protein extracts showed that the inhibition of mir-130a also resulted in the up-regulation of two other identified targets KIFAP3 and CYP27A1, although to a lesser extent, 2- and 0.4-fold, respectively (Supplementary Figure S7).Figure 5.

Bottom Line: MicroRNAs (miRNA) are a class of small RNA molecules that regulate numerous critical cellular processes and bind to partially complementary sequences resulting in down-regulation of their target genes.To enable the identification of biologically relevant miRNA targets, we describe a novel functional assay based on a 3'-UTR-enriched library and a positive/negative selection strategy.As proof of principle we have used mir-130a and its validated target MAFB to test this strategy.

View Article: PubMed Central - PubMed

Affiliation: Department of Haematological Medicine, King's College London, Rayne Institute, London SE5 9NU, UK. joop.gaken@kcl.ac.uk

ABSTRACT
MicroRNAs (miRNA) are a class of small RNA molecules that regulate numerous critical cellular processes and bind to partially complementary sequences resulting in down-regulation of their target genes. Due to the incomplete homology of the miRNA to its target site identification of miRNA target genes is difficult and currently based on computational algorithms predicting large numbers of potential targets for a given miRNA. To enable the identification of biologically relevant miRNA targets, we describe a novel functional assay based on a 3'-UTR-enriched library and a positive/negative selection strategy. As proof of principle we have used mir-130a and its validated target MAFB to test this strategy. Identification of MAFB and five additional targets and their subsequent confirmation as mir-130a targets by western blot analysis and knockdown experiments validates this strategy for the functional identification of miRNA targets.

Show MeSH