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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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Western blot and QRT–PCR analysis of identified mir-130a targets. Endogenous protein expression levels were determined in untransfected, empty vector (pBabepuro) and mir-130a transfected cells (pBabepuro-mir-130a). Cells were transduced using the BES precipitation procedure. (A) HepG2 cell extract probed with MAFB antibody (Santa Cruz) showing down-regulation of MAFB in mir-130a expressing cells. (B–D) MCF7 cells probed with TPT1 antibody (Abcam), CYP27A1 antibody (Abcam) and KIFAP3 antibody (PTG Cambridge). Expression of the respective proteins was down-regulated in the presence of mir-130a. Blots were stripped and re-probed with a γ-tubulin antibody (Santa Cruz) as a loading control. Expression levels were quantified by densitometry as shown in the corresponding bar graphs. (E) Determination of mRNA levels by QRT–PCR. To investigate whether the down-regulation of mir-130a targets was due to mRNA degradation or inhibition of translation, the mRNA levels of MAFB, TPT1, CYP27A1 and KIFAP3 were quantified by QRT–PCR in triplicate on untransfected cells (UN), empty vector (pBabepuro) transfected cells (C) and pBabepuro-mir130a transfected cells (130a). The results show that mRNA levels remain unchanged indicating that the protein knockdown is due to inhibition of translation.
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gks145-F4: Western blot and QRT–PCR analysis of identified mir-130a targets. Endogenous protein expression levels were determined in untransfected, empty vector (pBabepuro) and mir-130a transfected cells (pBabepuro-mir-130a). Cells were transduced using the BES precipitation procedure. (A) HepG2 cell extract probed with MAFB antibody (Santa Cruz) showing down-regulation of MAFB in mir-130a expressing cells. (B–D) MCF7 cells probed with TPT1 antibody (Abcam), CYP27A1 antibody (Abcam) and KIFAP3 antibody (PTG Cambridge). Expression of the respective proteins was down-regulated in the presence of mir-130a. Blots were stripped and re-probed with a γ-tubulin antibody (Santa Cruz) as a loading control. Expression levels were quantified by densitometry as shown in the corresponding bar graphs. (E) Determination of mRNA levels by QRT–PCR. To investigate whether the down-regulation of mir-130a targets was due to mRNA degradation or inhibition of translation, the mRNA levels of MAFB, TPT1, CYP27A1 and KIFAP3 were quantified by QRT–PCR in triplicate on untransfected cells (UN), empty vector (pBabepuro) transfected cells (C) and pBabepuro-mir130a transfected cells (130a). The results show that mRNA levels remain unchanged indicating that the protein knockdown is due to inhibition of translation.

Mentions: To validate the putative mir-130a targets, western blot analysis of endogenous protein expression was performed in MCF7 cells transfected with the empty vector pBabepuro or pBabepuro-mir-130a. Protein extracts obtained from MCF7 cells were probed with antibodies to TPT1 (Abcam), KIFAP3 (PTG Cambridge) and CYP27A1 (Abcam). MCF7 cells do not express endogenous MAFB, therefore, HepG2 cells (which do express MAFB) were transfected with pBabepuro or pBabepuro-mir-130a and protein extract was used to probe for MAFB (Santa Cruz). Blots were stripped and re-probed with a γ-tubulin antibody (Santa Cruz). There were no commercial antibodies available for PRR14 and MITD1. The results showed clear down-regulation of MAFB, TPT1, KIFAP3 and CYP27A1 (Figure 4A–D). To determine whether these proteins were also regulated at the mRNA level, we analysed total RNA from the cell lines by QRT–PCR and found no difference in the levels of mRNA for MAFB and the novel mir-130a targets identified in this study, indicating that mir-130a modulates expression of these proteins at the translational level (Figure 4E).Figure 4.


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)

Western blot and QRT–PCR analysis of identified mir-130a targets. Endogenous protein expression levels were determined in untransfected, empty vector (pBabepuro) and mir-130a transfected cells (pBabepuro-mir-130a). Cells were transduced using the BES precipitation procedure. (A) HepG2 cell extract probed with MAFB antibody (Santa Cruz) showing down-regulation of MAFB in mir-130a expressing cells. (B–D) MCF7 cells probed with TPT1 antibody (Abcam), CYP27A1 antibody (Abcam) and KIFAP3 antibody (PTG Cambridge). Expression of the respective proteins was down-regulated in the presence of mir-130a. Blots were stripped and re-probed with a γ-tubulin antibody (Santa Cruz) as a loading control. Expression levels were quantified by densitometry as shown in the corresponding bar graphs. (E) Determination of mRNA levels by QRT–PCR. To investigate whether the down-regulation of mir-130a targets was due to mRNA degradation or inhibition of translation, the mRNA levels of MAFB, TPT1, CYP27A1 and KIFAP3 were quantified by QRT–PCR in triplicate on untransfected cells (UN), empty vector (pBabepuro) transfected cells (C) and pBabepuro-mir130a transfected cells (130a). The results show that mRNA levels remain unchanged indicating that the protein knockdown is due to inhibition of translation.
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gks145-F4: Western blot and QRT–PCR analysis of identified mir-130a targets. Endogenous protein expression levels were determined in untransfected, empty vector (pBabepuro) and mir-130a transfected cells (pBabepuro-mir-130a). Cells were transduced using the BES precipitation procedure. (A) HepG2 cell extract probed with MAFB antibody (Santa Cruz) showing down-regulation of MAFB in mir-130a expressing cells. (B–D) MCF7 cells probed with TPT1 antibody (Abcam), CYP27A1 antibody (Abcam) and KIFAP3 antibody (PTG Cambridge). Expression of the respective proteins was down-regulated in the presence of mir-130a. Blots were stripped and re-probed with a γ-tubulin antibody (Santa Cruz) as a loading control. Expression levels were quantified by densitometry as shown in the corresponding bar graphs. (E) Determination of mRNA levels by QRT–PCR. To investigate whether the down-regulation of mir-130a targets was due to mRNA degradation or inhibition of translation, the mRNA levels of MAFB, TPT1, CYP27A1 and KIFAP3 were quantified by QRT–PCR in triplicate on untransfected cells (UN), empty vector (pBabepuro) transfected cells (C) and pBabepuro-mir130a transfected cells (130a). The results show that mRNA levels remain unchanged indicating that the protein knockdown is due to inhibition of translation.
Mentions: To validate the putative mir-130a targets, western blot analysis of endogenous protein expression was performed in MCF7 cells transfected with the empty vector pBabepuro or pBabepuro-mir-130a. Protein extracts obtained from MCF7 cells were probed with antibodies to TPT1 (Abcam), KIFAP3 (PTG Cambridge) and CYP27A1 (Abcam). MCF7 cells do not express endogenous MAFB, therefore, HepG2 cells (which do express MAFB) were transfected with pBabepuro or pBabepuro-mir-130a and protein extract was used to probe for MAFB (Santa Cruz). Blots were stripped and re-probed with a γ-tubulin antibody (Santa Cruz). There were no commercial antibodies available for PRR14 and MITD1. The results showed clear down-regulation of MAFB, TPT1, KIFAP3 and CYP27A1 (Figure 4A–D). To determine whether these proteins were also regulated at the mRNA level, we analysed total RNA from the cell lines by QRT–PCR and found no difference in the levels of mRNA for MAFB and the novel mir-130a targets identified in this study, indicating that mir-130a modulates expression of these proteins at the translational level (Figure 4E).Figure 4.

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