Improved chondrogenic differentiation of mesenchymal stem cells (MSCs) by genetic regulation is a potential method for regenerating articular cartilage. MiR-127-5p has been reported to promote cartilage differentiation of rat bone marrow MSCs (rMSCs); however, the regulatory mechanisms underlying hypoxia-stimulated chondrogenic differentiation remain unknown. rMSCs were induced to undergo chondrogenic differentiation under normoxic or hypoxic conditions. Expression of lncRNA DNM3OS, miR-127-5p, and GREM2 was detected by quantitative real-time PCR. Proteoglycans were detected by Alcian blue staining. Western blot assays were performed to examine the relative levels of GREM2 and chondrogenic differentiation related proteins. Luciferase reporter assays were performed to assess the association among DNM3OS, miR-127-5p, and GREM2. MiR-127-5p levels were upregulated, while DNM3OS and GREM2 levels were downregulated in rMSCs induced to undergo chondrogenic differentiation, and those changes were attenuated by hypoxic conditions (1% O2). Further in vitro experiments revealed that downregulation of miR-127-5p reduced the production of proteoglycans and expression of chondrogenic differentiation markers (COL1A1, COL2A1, SOX9, and ACAN) and osteo/chondrogenic markers (BMP-2, p-SMAD1/2). MiR-127-5p overexpression produced the opposite results in rMSCs induced to undergo chondrogenic differentiation under hypoxic conditions. GREM2 was found to be a direct target of miR-127-5p, which was suppressed in rMSCs undergoing chondrogenic differentiation. Moreover, DNM3OS could directly bind to miR-127-5p and inhibit chondrogenic differentiation of rMSCs via regulating GREM2. Our study revealed a novel molecular pathway (DNM3OS/miR-127-5p/GREM2) that may be involved in hypoxic chondrogenic differentiation.

中文翻译：

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LncRNA DNM3OS通过miR-127-5p调控GREM2抑制缺氧条件下大鼠间充质干细胞的早期软骨分化

通过基因调控改善间充质干细胞 (MSCs) 的软骨分化是再生关节软骨的潜在方法。据报道，MiR-127-5p 可促进大鼠骨髓间充质干细胞 (rMSCs) 的软骨分化；然而，缺氧刺激软骨分化的调控机制仍然未知。rMSCs 在常氧或缺氧条件下被诱导成软骨分化。通过实时定量 PCR 检测 lncRNA DNM3OS、miR-127-5p 和 GREM2 的表达。通过阿尔辛蓝染色检测蛋白多糖。进行蛋白质印迹分析以检查 GREM2 和软骨分化相关蛋白的相对水平。进行荧光素酶报告基因检测以评估 DNM3OS、miR-127-5p 和 GREM2 之间的关联。MiR-127-5p 水平上调，而 DNM3OS 和 GREM2 水平在诱导进行软骨分化的 rMSC 中下调，并且这些变化因缺氧条件（1% O2）而减弱。进一步的体外实验表明，miR-127-5p 的下调减少了蛋白多糖的产生和软骨分化标志物（COL1A1、COL2A1、SOX9 和 ACAN）和骨/软骨形成标志物（BMP-2、p-SMAD1/2）的表达. MiR-127-5p 过表达在 rMSCs 中产生相反的结果，诱导在缺氧条件下进行软骨分化。发现 GREM2 是 miR-127-5p 的直接靶标，其在经历软骨分化的 rMSC 中受到抑制。此外，DNM3OS 可以直接结合 miR-127-5p 并通过调节 GREM2 抑制 rMSCs 的软骨分化。