Satb2 expression in Foxc1-promoted osteogenic differentiation of MC3T3-E1 cells is negatively regulated by microRNA-103-3p

Abstract

The forkhead transcription factor C1 (Foxc1) is a cell-fate-determining factor that controls cranial bone development and osteogenic differentiation. Previously, it was demonstrated that various microRNAs (miRNAs) play important roles in osteogenesis and regulate the complex process of osteogenic differentiation. However, it remains unclear how miRNA expression changes during Foxc1-promoted osteogenic differentiation. In this study, we successfully overexpressed the Foxc1 gene in MC3T3-E1 cells and investigated the alterations in the miRNA expression profile on day 3 after osteogenic induction by using a miRNA microarray. Nine downregulated miRNAs and eight upregulated miRNAs were found to be differentially expressed. Among these miRNAs, miR-103-3p was consistently downregulated in the Foxc1-overexpressing MC3T3-E1 cells and was identified as a negative regulator of osteogenic differentiation by using a gain- and lose-of-function assay. The special AT-rich sequence-binding protein 2 (Satb2), a pivotal osteogenic transcription factor, was identified as the miR-103-3p targeting gene and was verified by real-time polymerase chain reaction, western blot analysis, and luciferase assay. Overexpression of miR-103-3p markedly inhibited the expression of Satb2 and attenuated Foxc1-promoted osteogenic differentiation. Taken together, our results elucidated the miRNA expression profiles of MC3T3-E1 cells in the early stage of Foxc1-promoted osteogenic differentiation and suggested that miR-103-3p acts as a negative regulator of the osteogenic differentiation of MC3T3-E1 cells by directly targeting Satb2.