Department of Histology and Embryology, School of Basic Medical Sciences, Capital Medical University, Beijing, China.
Musculosketetal Tissue Bank, Beijing Jishuitan Hospital, Beijing, China.
J Tissue Eng Regen Med. 2020 Nov;14(11):1618-1629. doi: 10.1002/term.3122. Epub 2020 Sep 9.
Mesenchymal stem cells (MSCs) are considered a promising candidate for use in cell-based therapy for cartilage repair. To promote understanding of the molecular control of chondrogenesis differentiation in MSCs, we compared the changes in microRNAs during in vitro chondrogenesis process of human bone-marrow mesenchymal stem cells (hBMSCs). MiR-199b-5p was up-regulated significantly during this process. The aim of the study was to investigate the effects of miR-199b-5p on chondrogenic differentiation of C3H10T1/2 MSC cells and explore the underlying mechanisms. MiR-199b-5p mimics or inhibitor were transfected into C3H10T1/2 cells, respectively, and then, the effects of miR-199b-5p on chondrogenic differentiation of C3H10T1/2 cells were detected. The results indicated that miR-199b-5p overexpression inhibited the growth of C3H10T1/2 cells but promoted transforming growth factor-β3 (TGF-β3)-induced C3H10T1/2 cells of chondrogenic differentiation, as supported by enhancing the gene and protein expression of chondrocyte specific markers of SOX9, aggrecan, and collagen type II (Col2a1). In contrast, inhibiting miR-199b-5p notably promoted the proliferation of C3H10T1/2 cells but decreased chondrogenic differentiation. Furthermore, mechanism studies revealed that JAG1 was a direct target of miR-199b-5p by dual luciferase reporter assays. While silencing of JAG1 by isRNA resulted an increase of chondrogenic differentiation. Further, JAG1 knockdown was demonstrated to block the effect of miR-199b-5p inhibition. In conclusion, the present study revealed for the first time that miR-199b-5p was the positive regulators to modulate chondrogenic differentiation of C3H10T1/2 cells by targeting JAG1. These findings may provide a novel insight on miRNA-mediated MSC therapy for cartilage related disorders.
间充质干细胞（MSCs）被认为是用于软骨修复的细胞治疗的有前途的候选物。为了促进对 MSCs 软骨形成分化的分子调控的理解，我们比较了人骨髓间充质干细胞（hBMSCs）体外软骨形成过程中 microRNAs 的变化。在此过程中，miR-199b-5p 显著上调。本研究旨在探讨 miR-199b-5p 对 C3H10T1/2 MSC 细胞软骨分化的影响，并探讨其潜在机制。分别将 miR-199b-5p 模拟物或抑制剂转染到 C3H10T1/2 细胞中，然后检测 miR-199b-5p 对 C3H10T1/2 细胞软骨分化的影响。结果表明，miR-199b-5p 过表达抑制 C3H10T1/2 细胞的生长，但促进转化生长因子-β3（TGF-β3）诱导的 C3H10T1/2 细胞的软骨分化，这一结果得到了软骨细胞特异性标志物 SOX9、聚集蛋白聚糖和胶原 II（Col2a1）基因和蛋白表达增强的支持。相反，抑制 miR-199b-5p 显著促进 C3H10T1/2 细胞的增殖，但减少软骨分化。此外，机制研究表明，双荧光素酶报告基因实验证实 JAG1 是 miR-199b-5p 的直接靶标。用 siRNA 沉默 JAG1 可增加软骨分化。此外，JAG1 敲低被证明可以阻断 miR-199b-5p 抑制的作用。综上所述，本研究首次揭示，miR-199b-5p 通过靶向 JAG1 作为正调节剂来调节 C3H10T1/2 细胞的软骨分化。这些发现可能为 miRNA 介导的 MSC 治疗软骨相关疾病提供新的见解。
