Wang Han, Fan Mingxing, An Yan, He Da
Department of Spine Surgery, Beijing Jishuitan Hospital, The Fourth Medical College of Peking University, Beijing, China.
Stem Cells Int. 2023 Jan 5;2023:7545635. doi: 10.1155/2023/7545635. eCollection 2023.
Bone marrow-derived mesenchymal stem cells (BMSCs) have a superior potential of osteogenic differentiation (OD) and a promising stem cell type to treat bone defects. This study sought to investigate the molecular mechanism of long noncoding RNA small nucleolar RNA host gene 14 (SNHG14) in OD of BMSCs. Western blot analysis or RT-qPCR showed that SNHG14, neural precursor cell expressed developmentally downregulated 4-like (NEDD4L), and Purkinje cell protein 4 (PCP4) were upregulated whereas forkhead box A2 (FOXA2) was declined in OD of BMSCs. RT-qPCR and cell staining showed that SNHG14 downregulation repressed OD of BMSCs, as manifested by reductions in osteopontin and osteocalcin levels, the mineralization degree, and alkaline phosphatase activity. RNA/Co/chromatin immunoprecipitation and dual-luciferase assays and determination of mRNA stability and ubiquitination level showed that SNHG14 bound to human antigen R improves NEDD4L mRNA stability and expression, further promoted FOXA2 ubiquitination to inhibit FOXA2 expression, and then reduced FOXA2 enrichment on the PCP4 promoter to upregulate PCP4 transcription. Functional rescue experiments showed that the overexpression of NEDD4L or PCP4 and knockdown of FOXA2 both attenuated the inhibition of SNHG14 downregulation on OD of BMSCs. Overall, our findings suggested that SNHG14 promoted OD of BMSCs through the NEDD4L/FOXA2/PCP4 axis.
骨髓间充质干细胞(BMSCs)具有卓越的成骨分化(OD)潜能,是治疗骨缺损颇具前景的干细胞类型。本研究旨在探究长链非编码RNA小核仁RNA宿主基因14(SNHG14)在BMSCs成骨分化中的分子机制。蛋白质免疫印迹分析或逆转录定量聚合酶链反应(RT-qPCR)结果显示,在BMSCs成骨分化过程中,SNHG14、神经前体细胞表达发育下调蛋白4样蛋白(NEDD4L)和浦肯野细胞蛋白4(PCP4)表达上调,而叉头框A2(FOXA2)表达下降。RT-qPCR和细胞染色结果表明,SNHG14表达下调抑制了BMSCs的成骨分化,表现为骨桥蛋白和骨钙素水平降低、矿化程度降低以及碱性磷酸酶活性降低。RNA/蛋白质/染色质免疫沉淀实验、双荧光素酶报告基因检测以及mRNA稳定性和泛素化水平测定结果表明,SNHG14与人抗原R结合可提高NEDD4L mRNA稳定性和表达水平,进一步促进FOXA2泛素化以抑制FOXA2表达,进而减少FOXA2在PCP4启动子上的富集,上调PCP4转录。功能挽救实验表明,过表达NEDD4L或PCP4以及敲低FOXA2均能减弱SNHG14表达下调对BMSCs成骨分化的抑制作用。总体而言,我们的研究结果表明,SNHG14通过NEDD4L/FOXA2/PCP4轴促进BMSCs的成骨分化。