Y-Y Gong1, M-Y Peng, D-Q Yin, Y-F Yang. 1. Department of Prosthodontics, Affiliated Stomatology Hospital of Chongqing Medical University, Chongqing Research Center for Oral Diseases and Biomedical Science, Chongqing, China. mryongfanyang@gmail.com.
Abstract
OBJECTIVE: To investigate the molecular mechanism of long noncoding RNA (lncRNA) H19 that promotes osteogenic differentiation in rat ectomesenchymal stem cells (EMSCs). MATERIALS AND METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.
OBJECTIVE: To investigate the molecular mechanism of long noncoding RNA (lncRNA) H19 that promotes osteogenic differentiation in rat ectomesenchymal stem cells (EMSCs). MATERIALS AND METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.