MiR-200b is involved in Tgf-β signaling to regulate mammalian palate development

Abstract

Various cellular and molecular events are involved in palatogenesis, including apoptosis, epithelial-mesenchymal transition (EMT), cell proliferation, and cell migration. Smad2 and Snail, which are well-known key mediators of the transforming growth factor beta (Tgf-β) pathway, play a crucial role in the regulation of palate development. Regulatory effects of microRNA 200b (miR-200b) on Smad2 and Snail in palatogenesis have not yet been elucidated. The aim of this study is to determine the relationship between palate development regulators miR-200b and Tgf-β-mediated genes. Expression of miR-200b, E-cadherin, Smad2, and Snail was detected in the mesenchyme of the mouse palate, while miR-200b was expressed in the medial edge epithelium (MEE) and palatal mesenchyme. After the contact of palatal shelves, miR-200b was no longer expressed in the mesenchyme around the fusion region. The binding activity of miR-200b to both Smad2 and Snail was examined using a luciferase assay. MiR-200b directly targeted Smad2 and Snail at both cellular and molecular levels. The function of miR-200b was determined by overexpression via a lentiviral vector in the palatal shelves. Ectopic expression of miR-200b resulted in suppression of these Tgf-β-mediated regulators and changes of apoptosis and cell proliferation in the palatal fusion region. These results suggest that miR-200b plays a crucial role in regulating the Smad2, Snail, and in apoptosis during palatogenesis by acting as a direct non-coding, influencing factor. Furthermore, the molecular interactions between miR-200b and Tgf-β signaling are important for proper palatogenesis and especially for palate fusion. Elucidating the mechanism of palatogenesis may aid the design of effective gene-based therapies for the treatment of congenital cleft palate.

Identification of the Differentially Expressed microRNAs Involved in Cleft Palate Induced by Retinoic Acid (RA) in Mouse Model

“…Palate development is a complex mechanism that involves elevation, contact, and fusion of palatal process 21) . Therefore, correct gene expression is necessary for proper palate development in both palatal epithelium and mesenchyme 22,23,24) . miRNAs microarray chip, as a kind of rapid and effi cient method to analyze the miRNAs expression profi les, has been widely used to identify the differentially expressed miRNAs in tumor 25,26) .…”

Epidermal Growth Factor Impairs Palatal Shelf Adhesion and Fusion in the <b><i>Tgf-β</i></b>₃ Null Mutant

“…Mouse Tgf-β 3 -/-MEE shows a great change in cell polarity compared to WT mice, with a persistent and distorted distribution of β-catenin in the cytoplasm and an abnormal presence of E-cadherin, α-catenin, β-actin, vinculin, laminin and the β2-integrin and its ligand intercellular adhesion molecule-1 in the most superficial apical surface of the MEE cells Martínez-Sanz et al, 2008]. Furthermore, Snail is expressed in Tgf-β 3 -/-mouse pre-adhesion MEE, which does not occur in WT mice [Martínez-Álvarez et al, 2004;Shin et al, 2012]. However, Tgf-β 3 -/-mouse MEE cells do not adhere, intercalate or transform into mesenchyme [Kaartinen et al, 1997] and do retain epithelial characteristics and eventually keratinise as epithelial cells from the oral region [Proetzel et al, 1995;Martínez-Álvarez et al, 2004].…”

“…It might also block repressors of the TGF-β signalling pathway, thus restoring cell death ad integrum. For example, the palate development regulator microRNA 200b can directly suppress the expression of Smad-2 in the MES of WT palate cultures, causing a reduction in MES cell death and an increase in cell proliferation [Shin et al, 2012].…”

8 Epidemiology of Cleft Lip and Palate

“…They are involved in gene silencing and play important roles in cell and tissue differentiation, including development of the secondary palate [40][41][42][43]. miRNAs have been shown to orchestrate many of the processes that are central to palatal morphogenesis, including epithelial-mesenchymal transformation, platelet-derived growth factor (PDGF) and TGF-β signaling, cell migration and proliferation, and collagen synthesis [44][45][46][47][48]. As such, further analysis of miRNA expression and gene networks will be key to elucidating mechanisms of palatal development as well as etiologies of OFC.…”