Mir‐29b promotes human aortic valve interstitial cell calcification via inhibiting TGF‐β3 through activation of wnt3/β‐catenin/Smad3 signaling

Herein, we hypothesized that pro-osteogenic MicroRNAs (miRs) could play functional roles in the calcification of the aortic valve and aimed to explore the functional role of miR-29b in the osteoblastic differentiation of human aortic valve interstitial cells (hAVICs) and the underlying molecular mechanism. Osteoblastic differentiation of hAVICs isolated from human calcific aortic valve leaflets obtained intraoperatively was induced with an osteogenic medium. Alizarin red S staining was used to evaluate calcium deposition. The protein levels of osteogenic markers and other proteins were evaluated using western blotting and/or immunofluorescence while qRT-PCR was applied for miR and mRNA determination. Bioinformatics and luciferase reporter assay were used to identify the possible interaction between miR-29b and TGF-β3. Calcium deposition and the number of calcification nodules were pointedly and progressively increased in hAVICs during osteogenic differentiation. The levels of osteogenic and calcification markers were equally increased, thus confirming the mineralization of hAVICs. The expression of miR-29b was significantly increased during osteoblastic differentiation. Furthermore, the osteoblastic differentiation of hAVICs was significantly inhibited by the miR-29b inhibition. TGF-β3 was markedly downregulated while Smad3, Runx2, wnt3, and β-catenin were significantly upregulated during osteogenic induction at both the mRNA and protein levels. These effects were systematically induced by miR-29b overexpression while the inhibition of miR-29b showed the inverse trends. Moreover, TGF-β3 was a direct target of miR-29b. Inhibition of miR-29b hinders valvular calcification through the upregulation of the TGF-β3 via inhibition of wnt/β-catenin and RUNX2/Smad3 signaling pathways.