A functional assay for microRNA target identification and validation

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.

Figure 1.

Schematic representation of the functional assay for miRNA target discovery.

Figure 2.

MCF7 cells were transfected with the plasmid library and selected in 500 μg/ml zeocin. Zeocin resistant and normal MCF7 cells were selected in different concentrations of GCV showing GCV sensitivity of the library transfected cells.

Figure 3.

GCV selection of library transfected cells with or without mir-130a. Cells (10⁶ MCF7 cells) transfected with p3′TKzeo + library (A) or MCF7 cells transfected with p3′TKzeo + library + pBabepuro empty vector (B) or transfected with p3′TKzeo + library + pBabepuro-mir-130a (C) were plated in 10-cm plates and selected in 8 μM GCV. After 10 days plates were stained with Coomassie blue.

Figure 4.

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.

Figure 5.

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.