LncRNA H19 Regulates BMP2-Induced Hypertrophic Differentiation of Mesenchymal Stem Cells by Promoting Runx2 Phosphorylation
LncRNA H19 Regulates BMP2-Induced Hypertrophic Differentiation of Mesenchymal Stem Cells by Promoting Runx2 Phosphorylation
复制标题
DOI：
10.3389/fcell.2020.00580
复制
发表时间：
2020-07-29
影响因子：
5.5
通讯作者：
Huang, Wei
中科院分区：
文献类型：
--
作者：
Dai, Guangming;Xiao, Haozhuo;Huang, Wei
目的骨形态发生蛋白2(BMP2)可诱导间充质干细胞(MSCs)向软骨方向分化，从而阻碍BMP2介导的软骨组织工程的进一步应用。在此，我们研究了BMP2介导的MSCs肥大分化的潜在机制。材料与方法体外构建骨髓间充质干细胞向活体软骨分化模型。用荧光原位杂交(FISH)技术检测H19在小鼠肢体中的表达。通过腺病毒载体表达转基因BMP2、H19沉默和过表达。采用逆转录定量实时聚合酶链式反应(RT-qPCR)、Western印迹和免疫组织化学方法检测基因表达。用Spearman相关系数计算H19表达与其他参数的相关性。通过RNA免疫沉淀(RIP)鉴定H19和Runx2的结合。结果H19在小鼠肢体中的表达水平在增殖区最高，从增生区到增生区逐渐降低。在BMP2刺激下，H19的表达水平最高，其次是Sox9的表达峰值；同时，H19的表达水平与软骨分化标志物呈正相关，尤其是在BMP2刺激的晚期，而与肥大分化标志物呈负相关。我们进一步的实验通过体外和活体实验发现，沉默H19促进了BMP2诱导的软骨细胞的肥大分化，这表明H19对于维持BMP2诱导的软骨细胞的表型具有重要作用。在机制上，我们研究了H19通过促进Runx2的磷酸化来调控BMP2介导的MSCs肥大分化。结论H19可能通过促进Runx2的磷酸化来调节BMP2诱导的MSCs肥大分化。
Objectives Bone morphogenetic protein 2 (BMP2) triggers hypertrophic differentiation after chondrogenic differentiation of mesenchymal stem cells (MSCs), which blocked the further application of BMP2-mediated cartilage tissue engineering. Here, we investigated the underlying mechanisms of BMP2-mediated hypertrophic differentiation of MSCs. Materials and Methods In vitroandin vivochondrogenic differentiation models of MSCs were constructed. The expression of H19 in mouse limb was detected by fluorescencein situhybridization (FISH) analysis. Transgenes BMP2, H19 silencing, and overexpression were expressed by adenoviral vectors. Gene expression was determined by reverse transcription and quantitative real-time PCR (RT-qPCR), Western blot, and immunohistochemistry. Correlations between H19 expressions and other parameters were calculated with Spearman's correlation coefficients. The combination of H19 and Runx2 was identified by RNA immunoprecipitation (RIP) analysis. Results We identified that H19 expression level was highest in proliferative zone and decreased gradually from prehypertrophic zone to hypertrophic zone in mouse limbs. With the stimulation of BMP2, the highest expression level of H19 was followed after the peak expression level of Sox9; meanwhile, H19 expression levels were positively correlated with chondrogenic differentiation markers, especially in the late stage of BMP2 stimulation, and negatively correlated with hypertrophic differentiation markers. Our further experiments found that silencing H19 promoted BMP2-triggered hypertrophic differentiation throughin vitroandin vivotests, which indicated the essential role of H19 for maintaining the phenotype of BMP2-induced chondrocytes. In mechanism, we characterized that H19 regulated BMP2-mediated hypertrophic differentiation of MSCs by promoting the phosphorylation of Runx2. Conclusion These findings suggested that H19 regulates BMP2-induced hypertrophic differentiation of MSCs by promoting the phosphorylation of Runx2.