AbstractBackgroundAll-trans retinoic acid (atRA) results in cleft palate, but the cellular and molecular mechanisms underlying the teratogenic effects on palatal development have not been fully elucidated. Autophagy interruption has been reported to seriously affect embryonic-cell differentiation and development. This study aimed to verify whether atRA-induced cleft palate occurs because atRA blocks autophagy and stemness of embryonic palatal mesenchyme (MEPM) cells, which are maintained via the phosphatase and tensin homolog (PTEN)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) autophagic signaling pathway, and inhibits osteogenic-differentiation potential of MEPM cells, which could lead to the development of cleft palate.MethodsTo assess the stemness and pluripotency of MEPM cells, we analyzed their surfacemarkers using immunofluorescence (IF) and flow cytometry (FCM). Differentiation potentials, such as osteogenic, adipogenic, and chondrogenic differentiation, were induced. We also investigated the role of the PTEN/Akt/mTOR autophagic signaling pathway, which maintains the stemness and pluripotency of MEPM cells. Using transmission electron microscopy (TEM), Western blot analysis, quantitative reverse transcriptase polymerase chain reaction (RT-qPCR), messenger ribonucleic acid (mRNA) microarray, dual-luciferase reporter system, and exosomes, we found that atRA blocks autophagy and osteogenic differentiation of MEPM cells through micro-ribonucleic acid (miR)-106a-5p by targeting the PTEN/Akt/mTOR autophagic pathway.ResultsIn vitro purified MEPM cells expressed cell surface markers similar to those of mouse bone marrow stem cells. Additionally, in vitro MEPM cells were ectomesenchymal and expressed the neural-crest marker human natural killer-1 (HNK-1), the mesodermal marker vimentin, and the ectodermal marker nestin. They were also positive for in vitro MEPM markers, including platelet-derived growth factor alpha (PDGFRα), ephrin B1 (Efnb1), odd-skipped related 2 (Osr2), and Meox2, as well as for stemness markers including POU class 5 homeobox 4 (Oct4), Nanog, and sex-determining region Y-related HMG box 2 (Sox2). MEPM cell pluripotency was retained through activation of the PTEN/Akt/mTOR autophagic signaling pathway. We found that atRA blocked MEPM cell pluripotency to inhibit osteogenic differentiation via miR-106a-5p targeting of PTEN mRNA and subsequent suppression of the PTEN/Akt/mTOR autophagic pathway.ConclusionsIn vitro cultured MEPM cells are ectomesenchymal stem cells that have strong osteogenic differentiation potential, and MEPM pluripotency is regulated by autophagy via the PTEN/AKT/mTOR signaling pathway. atRA disrupts MEPM cell pluripotency through PTEN/AKT/mTOR signaling inactivation where miR-106a-5p targets PTEN mRNA to reduce osteogenic differentiation of MEPM cells and results in the development of cleft palates. Our findings provide new insight into the mechanism underlying the development of cleft palate, and miR-106a-5p may act as a prenatal screening biomarker for cleft palate as well as a new diagnostic and therapeutic target.
All-trans Retinoic Acid Regulates miR-106a-5p Inhibition of Autophagic in Developing Cleft Palates
