Genome-Wide mRNA-Seq Profiling Reveals that LEF1 and SMAD3 Regulate Epithelial-Mesenchymal Transition Through the Hippo Signaling Pathway During Palatal Fusion.

ABSTRACT

BACKGROUND: Epithelial-mesenchymal transition (EMT) of the medial edge epithelium (MEE) occurs through fusion of the palatal shelves and is a crucial step in palatogenesis. The key genes, however, and the related signaling pathway of EMT are not yet fully understood. Therefore, the aim of this study was to reveal the key genes and the related signaling pathway of EMT during palatal fusion. MATERIALS AND METHODS: C57BL/6J mice at embryonic gestation day 14.5 (E14.5; n = 6) were used to establish the cleft palate model for mRNA-Seq (HiSeq X Ten). The Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed for functional annotations of the differentially expressed genes. Quantitative polymerase chain reaction (qPCR) assays were used to validate the RNAseq data. RESULTS: A total of 936 differentially expressed genes, including 558 upregulated and 378 downregulated genes were identified in cases versus controls, respectively. Among these genes, the GO analysis showed that Lymphoid Enhancer-Binding Factor 1 (LEF1) and SMAD Family Member 3 (SMAD3) significantly enriched biological processes, which were EMT related. The KEGG analysis showed that these genes regulated EMT through the Hippo signaling pathway. LEF1 and SMAD3 were downregulated, and the qPCR results corroborated the RNA-seq data. CONCLUSIONS: These results demonstrate that LEF1 and SMAD3 inhibits EMT at the MEE through the Hippo signaling pathway; and that this could contribute to cleft palate formation in embryonic palatal fusion at E 14.5.