Figure 5

RUNX2 mRNA is a primary target for miR-30a and miR-30d. (a) Predicted interaction between miR-30a and miR-30d and their putative binding sites in the 3' UTR of RUNX2. The representation is limited to the region around the miR-30a and miR-30d complementary sites. In bold is the 'seed' region with a conserved anchoring adenosine that is complementary to the first nucleotide of miR-30a and miR-30d (underlined). (b) Schematic representation of the construct used in the luciferase assay: a 300-bp (reporter 1) and 319-bp (reporter 2) region of the 3' UTR of human RUNX2 containing the putative miR-30a and/or miR-30d target sites (black boxes) were cloned into the pSi-CHECK™-2 vector. (c) Normalized luciferase activity 48 hours after co-transfection of human pre-miR-30a, pre-miR-30d, pre-miR-378 or pre-miR-control (neg) together with pSi-CHECK™-2 constructs in HEK 293 cells. Data were obtained from four independent experiments (error bars represent average ± standard error); n.s., not significant compared to pre-miR-control; *significant compared to pre-miR-control (P < 0.05); **significant compared to pre-miR-control (P < 0.01). (d) Undifferentiated hMADS cells were transfected with either pre-miR control (pre-miR-neg) or pre-miR-30a or pre-miR-30d. Four days later, cell lysates were prepared and expression of RUNX2 was investigated by western blotting. Tubulin was used as a loading control. The integrated density of each band was quantified with Image J. Densities obtained for RUNX2 signals were divided by the corresponding tubulin densities. Numbers below the blot are the density fold changes compared to the control condition. (e) Undifferentiated hMADS cells were transfected with control target site blocker (TSB-neg) or with RUNX2 target site blocker (TSB-RUNX2) as well as with pre-miR-30a. Adipogenic differentiation was evaluated by analyzing adiponectin and PPARγ expression by qPCR. The level of expression of each gene in the pre-miR-30a condition was taken as 1.