环状 RNA 0005736 通过 miR-636/MAPK1 轴促进肌腱源性干细胞的成肌腱分化。
Circ_0005736 promotes tenogenic differentiation of tendon-derived stem cells through the miR-636/MAPK1 axis.
机构信息
Department of Sports, Communication University of China, Nanjing, No.26, Pengshan Road, Jiangning District, Nanjing, 211172, China.
Faculty of Cultural Management, Communication University of China, Nanjing, Nanjing, China.
出版信息
J Orthop Surg Res. 2023 Sep 5;18(1):660. doi: 10.1186/s13018-023-04115-7.
BACKGROUND
Tendon-derived stem cells (TDSCs) are one of stem cells characterized by greater clonogenicity, tenogenesis, and proliferation capacity. Circ_0005736 has been shown to be decreased in Rotator cuff tendinopathy. Here, we investigated the function and relationship of circ_0005736 in TDSC tenogenic differentiation.
METHODS
Transforming growth factor β1 (TGF-β1) was used to induce the tenogenic differentiation in TDSC. Cell proliferation, invasion and migration were evaluated by Cell Counting Kit-8, 5-Ethynyl-2'-deoxyuridine, transwell, and wound healing assays, respectively. The detection of the levels of genes and proteins was performed by qRT-PCR and Western blot. The binding between miR-636 and circ_0005736 or MAPK1 (Mitogen-Activated Protein Kinase 1) was verified using dual-luciferase reporter assay and RIP assays.
RESULTS
TGF-β1 induced tenogenic differentiation by enhancing the production of tendon-specific markers and TDSC proliferation, invasion and migration. TGF-β1 treatment promoted circ_0005736 expression, knockdown of circ_0005736 abolished TGF-β1-induced tenogenic differentiation in TDSCs. Mechanistically, circ_0005736 acted as a sponge for miR-636 to up-regulate the expression of MAPK1, which was confirmed to be a target of miR-636 in TDSCs. Further rescue assays showed that inhibition of miR-636 could rescue circ_0005736 knockdown-induced suppression on TGF-β1-caused tenogenic differentiation in TDSCs. Moreover, forced expression of miR-636 abolished TGF-β1-caused tenogenic differentiation in TDSCs, which was rescued by MAPK1 up-regulation.
CONCLUSION
Circ_0005736 enhanced TGF-β1-induced tenogenic differentiation in TDSCs via increasing the production of tendon-specific markers and TDSC proliferation, invasion and migration through miR-636/MAPK1 axis.
背景
肌腱衍生干细胞（TDSCs）是一种具有更高克隆性、肌腱生成能力和增殖能力的干细胞。已经表明，环状 RNA 0005736 在肩袖肌腱病中减少。在这里，我们研究了 circ_0005736 在 TDSC 肌腱生成分化中的功能和关系。
方法
转化生长因子 β1（TGF-β1）用于诱导 TDSC 的肌腱生成分化。通过细胞计数试剂盒-8、5-乙炔基-2'-脱氧尿苷、transwell 和划痕愈合试验分别评估细胞增殖、侵袭和迁移。通过 qRT-PCR 和 Western blot 检测基因和蛋白水平。通过双荧光素酶报告基因检测和 RIP 试验验证 miR-636 与 circ_0005736 或 MAPK1（丝裂原活化蛋白激酶 1）之间的结合。
结果
TGF-β1 通过增强肌腱特异性标志物的产生和 TDSC 的增殖、侵袭和迁移来诱导肌腱生成分化。TGF-β1 处理促进 circ_0005736 的表达，circ_0005736 的敲低消除了 TDSC 中的 TGF-β1 诱导的肌腱生成分化。机制上，circ_0005736 作为 miR-636 的海绵，上调 MAPK1 的表达，这在 TDSC 中被证实是 miR-636 的靶标。进一步的挽救试验表明，抑制 miR-636 可以挽救 circ_0005736 敲低对 TDSC 中 TGF-β1 引起的肌腱生成分化的抑制作用。此外，miR-636 的强制表达消除了 TDSC 中 TGF-β1 引起的肌腱生成分化，而 MAPK1 的上调挽救了这一作用。
结论
circ_0005736 通过增加肌腱特异性标志物的产生和 TDSC 的增殖、侵袭和迁移，增强 TGF-β1 诱导的 TDSC 肌腱生成分化，通过 miR-636/MAPK1 轴。
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