Long non-coding RNA H19 promotes the osteogenic differentiation of rat ectomesenchymal stem cells via Wnt/β-catenin signaling pathway.

Abstract

To investigate the molecular mechanism of long noncoding RNA (lncRNA) H19 that promotes osteogenic differentiation in rat ectomesenchymal stem cells (EMSCs). EMSCs were isolated from rat fetal facial processes by flow cytometry. Osteogenic markers CD29, CD90, CD44, CD57, Nestin and sox10 were detected by fluorescent immunoassay. β-catenin and Wnt pathway target genes were detected by Real-time polymerase chain reaction (RT-PCR) and Western blot after the construction of transient interference H19, a stable expression of H19 EMSCs cell line and the induction of osteogenic differentiation of EMSCs cells. EMSCs of H19 overexpression were treated with Wnt/beta-catenin signaling pathway inhibitor Wnt-C59, and the expressions of beta-catenin and osteogenic markers were detected by RT-PCR and Western blot. Furthermore, the mechanism of H19 regulating Wnt/beta-catenin signaling pathway was explored by transfecting miR-22 and miR-141 mimics and luciferase reporter assays. EMSCs were successfully isolated and identified, osteogenic markers CD29, CD90, CD44, CD57, Nestin and sox10 were significantly overexpressed. Osteogenesis-induced solution significantly increased the expression of H19 and osteogenic markers ALP, Runx2, BMP and OCN in EMSCs (p<0.05). Interference with H19 significantly inhibited the expressions of osteogenic markers, beta-catenin and target genes of Wnt/beta-catenin signaling pathway (p<0.05), while upregulation of H19 significantly promoted the expressions of these markers and genes in EMSCs (p<0.05). Wnt-C59 inhibitors treatment inhibited the Wnt/beta-catenin signaling pathway and osteogenic differentiation in EMSCs with H19 overexpression (p<0.05). Furthermore, H19 could block the inhibitory effect of miR-22 and miR-141 on β-catenin and activate the Wnt/beta-catenin signaling pathway after transfecting miR-22 mimics and miR-141 mimics in EMSCs (p<0.05). LncRNA H19 can promote the osteogenic differentiation of rat EMSCs by activating Wnt/beta-catenin signal, providing a theoretical basis for the application of EMSCs in tooth tissue engineering regeneration and repair.