Human bone marrow-derived mesenchymal stem cells (hBMSCs) have chondrocyte differentiation potential and are considered to be a cell source for cell-transplantation-mediated repair of cartilage defects, including those associated with osteoarthritis (OA). However, chondrocyte hypertrophic differentiation is a major obstacle for the application of hBMSCs in articular cartilage defect treatment. We have previously shown that microRNA-27b (miR-27b) inhibits hypertrophy of chondrocytes from rat knee cartilage. In this study, we investigated the role of miR-27b in chondrocyte hypertrophic differentiation of hBMSCs. Chondrogenic marker and microRNA expression in hBMSC chondrogenic pellets were evaluated using RT-qPCR and immunohistochemistry. The hBMSCs were transfected with miR-27b before inducing differentiation. Gene and protein expression levels were analyzed using RT-qPCR and western blot. Coimmunoprecipitation was used to confirm interaction between CBFB and RUNX2. Luciferase reporter assays were used to demonstrate that CBFB is a miR-27b target. Chondrogenic differentiation was evaluated in hBMSCs treated with shRNA targeting CBFB. Chondrogenic hBMSC pellets overexpressing miR-27b were implanted into cartilage lesions in model rats; therapeutic effects were assessed based on histology and immunohistochemistry. The hBMSCs showed typical MSC differentiation potentials. During chondrogenic differentiation, collagen 2 and 10 (COL2 and COL10), SOX9, and RUNX2 expression was upregulated. Expression of miR-140, miR-143, and miR-181a increased over time, whereas miR-27b and miR-221 were downregulated. Cartilage derived from hBMSC and overexpressing miR-27b exhibited higher expression of COL2 and SOX9, but lower expression of COL10, RUNX2, and CBFB than did the control cartilage. CBFB and RUNX2 formed a complex, and CBFB was identified as a novel miR-27b target. CBFB knockdown by shRNA during hBMSC chondrogenic differentiation led to significantly increased COL2 and SOX9 expression and decreased COL10 expression. Finally, miR-27b-overexpressing hBMSC chondrogenic pellets had better hyaline cartilage morphology and reduced expression of hypertrophic markers and tend to increase repair efficacy in vivo. MiR-27b plays an important role in preventing hypertrophic chondrogenesis of hBMSCs by targeting CBFB and is essential for maintaining a hyaline cartilage state. This study provides new insights into the mechanism of hBMSC chondrocyte differentiation and will aid in the development of strategies for treating cartilage injury based on hBMSC transplantation.

中文翻译：

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MicroRNA-27b靶向CBFB，以抑制人骨髓间充质干细胞向肥大软骨细胞的分化。

人骨髓来源的间充质干细胞（hBMSC）具有软骨细胞分化潜能，被认为是细胞移植介导的软骨缺损修复的细胞来源，包括与骨关节炎（OA）相关的缺损。然而，软骨细胞肥大分化是hBMSCs在关节软骨缺损治疗中的主要障碍。先前我们已经表明，microRNA-27b（miR-27b）抑制了来自大鼠膝盖软骨的软骨细胞肥大。在这项研究中，我们调查了miR-27b在hBMSC软骨细胞肥大分化中的作用。使用RT-qPCR和免疫组织化学评估hBMSC软骨形成沉淀中的软骨生成标记和microRNA表达。在诱导分化之前，用miR-27b转染了hBMSC。使用RT-qPCR和Western blot分析基因和蛋白质表达水平。使用共免疫沉淀法来确认CBFB和RUNX2之间的相互作用。萤光素酶报告基因测定用于证明CBFB是miR-27b靶标。在靶向CBFB的shRNA处理的hBMSC中评估了软骨分化。将过表达miR-27b的软骨源性hBMSC沉淀物植入模型大鼠的软骨损伤中；根据组织学和免疫组织化学评估治疗效果。hBMSCs显示出典型的MSC分化潜能。在软骨分化过程中，胶原2和10（COL2和COL10），SOX9和RUNX2的表达上调。随着时间的推移，miR-140，miR-143和miR-181a的表达增加，而miR-27b和miR-221的表达下调。与对照软骨相比，源自hBMSC且过表达miR-27b的软骨表现出更高的COL2和SOX9表达，但表现出更低的COL10，RUNX2和CBFB表达。CBFB和RUNX2形成复合物，CBFB被鉴定为新型miR-27b靶标。在hBMSC软骨分化过程中，shRNA对CBFB的抑制作用导致COL2和SOX9表达显着增加，而COL10表达下降。最后，miR-27b过表达的hBMSC软骨生成沉淀物具有更好的透明软骨形态，并减少了肥大标志物的表达，并倾向于提高体内修复效果。MiR-27b通过靶向CBFB在预防hBMSC肥大软骨形成中起重要作用，并且对于维持透明软骨状态至关重要。