Analyzing mRNA expression identifies Smad3 as a microRNA-140 target regulated only at protein level.

mRNA profiling is routinely used to identify microRNA targets, however, this high-throughput technology is not suitable for identifying targets regulated only at protein level. Here, we have developed and validated a novel methodology based on computational analysis of promoter sequences combined with mRNA microarray experiments to reveal transcription factors that are direct microRNA targets at the protein level. Using this approach we identified Smad3, a key transcription factor in the TGFbeta signaling pathway, as a direct miR-140 target. We showed that miR-140 suppressed the TGFbeta pathway through repression of Smad3 and that TGFbeta suppressed the accumulation of miR-140 forming a double negative feedback loop. Our findings establish a valid strategy for the discovery of microRNA targets regulated only at protein level, and we propose that additional targets could be identified by re-analysis of existing microarray datasets.