Abstract 15230: MiR-30e Reduces Osteogenic Differentiation of Bone Marrow Derived Mesenchymal Stem Cells by Repressing IGF2 Expression
Objective: In atherosclerosis, activation of an osteogenic transcriptional program in de-differentiated mesenchymal-like smooth muscle cells contributes substantially to the initiation of aortic calcification. MiR-30e was recently reported as the most downregulated miR in the aortas of atherogenic APOE-/- mice. To assess a causal link of deficient miR-30e, we tested the hypothesis that overexpression of miR-30e would inhibit osteogenic differentiation of bone marrow derived mesenchymal stem cells (MSCs).
Method and Results: Using qPCR and Elisa respectively, we confirmed that the expression of the mRNA and protein for the osteogenic marker osteopontin (OPN) was upregulated (p<.01) in aortas of 6 mo old APOE-/- mice (n=9) relative to wild type controls (n=4). In MSCs transduced with lentivirus for miR-30e or a scrambled (scr) control, microarrays and qPCR showed repression (p<.05) of an osteogenic profile including Opn, Igf2, Runx2, Bmp4, Dpt, and Dcn genes. At 2 wk osteogenic differentiation, this osteogenic gene expression panel was dramatically enhanced in the MSCs but reduced (p<.05) by miR-30e, with a decrease (p<.05) in OPN protein levels. Secreted IGF2 protein was decreased in the MSCs over-expressing miR-30e at 1wk osteogenic differentiation. Electron microscopy after 2wk osteogenic differentiation showed a marked morphological difference in the miR-30e stables versus controls. MiR-30e oligos with 2’Fluoro-modification also significantly decreased the above osteogenic panel together with Alp1 transcripts, in a dose-dependent manner. MiR-30e reduced (p<.01) proliferation of MSCs at Days 2, 4, and 5 post plating. Inhibiting miR-30e in MSCs using antimiR-30e oligos followed by microarrays and qPCR showed over-expression of Igf2 transcripts suggesting that Igf2 could be a primary target of miR-30e-driven osteogenic repression. Double luciferase assay showed binding of miR-30e to the 3’UTR of Igf2 (p=.00001) and positive control Runx2 (p=.01) but not Opn.
Conclusion: miR-30e represses the osteogenic potential of MSCs by targeting IGF2, and its dowregulation may be a proximate cause of the initiation of atherosclerosis. The miR-30e pathway represents a potentially powerful and important clinical target in vascular diseases.
- © 2013 by American Heart Association, Inc.