These genes showed consistent profiles after inactivation of the miR 30 family and over expression of miR 30a. miR 30 miRNAs stimulate promotion information adipogenesis via inhibition of the osteogenesis transcription factor RUNX2 To identify molecular mechanisms that would regulate the effects of miR 30 miRNAs on adipogenesis, bioinfor matics prediction of their targets was performed with TargetScan. It revealed that RUNX2 bears several conserved binding sites for these miRNAs. RUNX2, also known as CBFA1, is a key regulator of osteogenesis and its expression is detected Inhibitors,Modulators,Libraries at the undifferentiated state. It increases during osteogenesis and decreases during adipogenesis. In order to test whether RUNX2 is targeted by the miR 30 family, we cloned two regions of its 3 UTR that contain the predicted miR 30 binding sites into the pSi CHECK 2 vector, downstream of the Renilla transla tional stop codon.

The first region covers positions 32 to 332 of the RUNX2 3 UTR and contains a poorly vertebrate conserved putative miR 30 binding site. The second region covers positions 3,102 to 3,421 of the RUNX2 3 UTR and encompasses two vertebrate Inhibitors,Modulators,Libraries con served putative binding sites. HEK 293T cells were co transfected with either con struct together with the following Inhibitors,Modulators,Libraries synthetic pre miRNAs negative control, miR 30a, miR 30d or miR 378. When cells were transfected with pSi CHECK 2 bearing the first putative binding site, none of the tested miRNAs had any effect on luciferase activity. In contrast, with pSi CHECK 2 bearing the last two binding sites, miR 30a and miR 30d triggered a more than two fold decrease in luciferase activity compared to the control miRNA.

As expected, miR 378 had no effect on luciferase Inhibitors,Modulators,Libraries activity. Importantly, this effect was confirmed at the pro tein level for endogenous Inhibitors,Modulators,Libraries RUNX2. Transfection of sub confluent hMADS cells with pre miR 30a or pre miR 30d induced a 0. 61 fold or 0. 48 fold decrease in RUNX2 pro tein levels, respectively. Thus, these results demonstrate that RUNX2 is a bona fide target of miR 30a and miR 30d. Finally, we sought to establish a direct link between miR 30 effects on adipogenesis and RUNX2 targeting. We used the target site blocker strategy to mask miR 30 binding sites 2 and 3 in the RUNX2 3 UTR. Transfection with RUNX2 miR 30 specific TSB, but not a control TSB, significantly decreased miR 30a stimula tion of adipogenesis.

download the handbook In conclusion, RUNX2 targeting is, at least in part, responsible for miR 30 posi tive effects on adipocyte differentiation. Discussion Adipocyte differentiation is a complex process combining several levels of regulation. Signaling pathways, such as cAMP and insulin signaling pathways, as well as key tran scription factors, such as PPARg, CEBPb and Kr��ppel like transcription factors, have been extensively studied. Our results suggest a direct role of miRNA mediated post transcriptional regulation in adipogenesis.