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

Abstract

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 TGFb signaling pathway, as a direct miR-140 target. We showed that miR-140 suppressed the TGFb pathway through repression of Smad3 and that TGFb 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.

MicroRNAs and post-transcriptional regulation of skeletal development

“…Of all the miRNAs affecting chondroblast differentiation, miR-140 has received the most research attention to date (He et al 2009, Nakamura et al 2011, Gibson & Asahara 2013, Karlsen et al 2013, Papaioannou et al 2013). The results of these studies indicate that miR-140 is one of the main regulators of chondroblast differentiation through its effects on the expression of not only Sox9 (Karlsen et al 2013), but also several other targets (Hdac4, Sp1, Smad3, and aggrecan) (Pais et al 2010, Nakamura et al 2011, Karlsen et al 2013. Moreover, independent groups have developed miR-140-null mice, which displayed a concordant phenotype with major growth defects of endochondral bones (Miyaki et al 2010, Nakamura et al 2011, Papaioannou et al 2013.…”

Significance of Epigenetic Landscape in Cartilage Regeneration from the Cartilage Development and Pathology Perspective

“…ACVR2B, SMAD2, and CHRDL1 were direct targets of miR-455-3p, which can also regulate TGF-b signaling during chondrogenesis [62]. Smad3 was identified as a direct target of miR-140; TGF-b signaling can be inhibited by miR-140 through suppression of Smad3 [63]. The set of miRNAs, including miR-221, miR-222, miR-140, miR-143, and miR-145, was enriched in the artificial zone of articular cartilage and exhibited expression changes with zonal differentiation.…”

A mikro-RNS-ek szerepe az agyalapimirigy-daganatok tumorbiológiájában

“…Több olyan miRNS került azonosításra, amelyek lehetséges célgénje a SMAD3 (1. táblázat) [42]. Ezek közül a miR-140-et egér-fibroblastsejtben már korábban validálták [68]. Valószínűsíthető tehát, hogy a nem funkcionáló adenomákban a miRNS-ek többek között a SMAD3 gátlásán keresztül megváltoztatják a TGFβ jelát-viteli útvonal működését, aminek szerepe lehet az adenomák patogenezisében.…”