Dai Guangming, Xiao Haozhuo, Zhao Chen, Chen Hong, Liao Junyi, Huang Wei
Department of Orthopaedic Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China.
Front Cell Dev Biol. 2020 Jul 29;8:580. doi: 10.3389/fcell.2020.00580. eCollection 2020.
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.
and chondrogenic differentiation models of MSCs were constructed. The expression of H19 in mouse limb was detected by fluorescence hybridization (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.
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 through and tests, 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.
These findings suggested that H19 regulates BMP2-induced hypertrophic differentiation of MSCs by promoting the phosphorylation of Runx2.
骨形态发生蛋白2（BMP2）可触发间充质干细胞（MSC）软骨形成分化后的肥大分化，这阻碍了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MSC肥大分化的潜在机制。
构建了MSC的软骨形成分化模型。通过荧光原位杂交（FISH）分析检测小鼠肢体中H19的表达。通过腺病毒载体表达转基因BMP2、H19沉默和过表达。通过逆转录和定量实时PCR（RT-qPCR）、蛋白质免疫印迹和免疫组织化学测定基因表达。用Spearman相关系数计算H19表达与其他参数之间的相关性。通过RNA免疫沉淀（RIP）分析鉴定H19与Runx2的结合。
我们发现H19表达水平在小鼠肢体的增殖区最高，从肥大前期区到肥大区逐渐降低。在BMP2刺激下，H19的最高表达水平在Sox9的峰值表达水平之后；同时，H19表达水平与软骨形成分化标志物呈正相关，尤其是在BMP2刺激后期，与肥大分化标志物呈负相关。我们的进一步实验发现，通过功能获得和功能缺失试验沉默H19可促进BMP2触发的肥大分化，这表明H19对维持BMP2诱导的软骨细胞表型至关重要。在机制上，我们发现H19通过促进Runx2的磷酸化来调节BMP2介导的MSC肥大分化。
这些发现表明，H19通过促进Runx2的磷酸化来调节BMP2诱导的MSC肥大分化。
