Zhang Qiang, Shen Yifei, Zhao Shujie, Jiang Yuqing, Zhou Dong, Zhang Yunkun
Department of Orthopedics, The Affiliated Changzhou No. 2 People's Hospital, Nanjing Medical University, Changzhou, Jiangsu 213003, China.
Department of Orthopedics, The People's Hospital of Jiangsu Province, Nanjing, Jiangsu 210029, China.
Cell Signal. 2021 Oct;86:110083. doi: 10.1016/j.cellsig.2021.110083. Epub 2021 Jul 10.
The physiology of the nucleus pulposus (NP) in intervertebral disc degeneration (IVD) has been studied widely. However, interactions involving nucleus pulposus -mesenchymal stem cells (NP-MSCs) are less understood. MicroRNA 15a (miR-15a) is known to target and modulate genes involved in cellular proliferation and apoptosis. This study aimed to understand the interactions and impact of miR-15a and NP-MSCs on chondrogenic differentiation and IVD degeneration. Exosomes secreted by NP cells were purified by differential centrifugation and identified by transmission electron microscopy and exosomal markers. Further, by co-culture these exosomes were re-introduced into the NP-MSC cells, which were confirmed by fluorescence confocal microscopy. NP-MSCs treated with exo-miR-15a increases aggrecan and collagen II mRNA and protein levels while decreasing mRNA and protein levels of ADAMTS4/5 and MMP-3/-13. Toluidine blue staining confirmed that chondrogenic differentiation was increased in NP-MSCs treated with exo-miR-15a. NP-MSCs treated with exo-anti-miR-15a inhibit aggrecan and collagen II expression while increasing ADAMTS4/5 and MMP-3/-13 expression and decreasing chondrogenic differentiation. Dual-luciferase reporter assays revealed that miR-15a directly targets MMP-3 and downregulates its expression. Overexpression of miR-15a increased proliferation and colony formation, whereas combinatorial overexpression with MMP3, suppressed miR-15a's effects. This was also evident through the decreased phosphorylation of PI3K and Akt, upregulation of Wnt3a and β-catenin in the presence of miR-15a, but overexpression of MMP3 indicated an opposite effect. Overall, these data demonstrate that exo-miR-15a promotes NP-MSCs chondrogenic differentiation by downregulating MMP-3 through PI3K/Akt and Wnt3a/β-catenin axis.
椎间盘退变（IVD）中髓核（NP）的生理学已得到广泛研究。然而，涉及髓核-间充质干细胞（NP-MSCs）的相互作用却鲜为人知。已知微小RNA 15a（miR-15a）靶向并调节参与细胞增殖和凋亡的基因。本研究旨在了解miR-15a与NP-MSCs之间的相互作用及其对软骨生成分化和IVD退变的影响。通过差速离心法纯化NP细胞分泌的外泌体，并通过透射电子显微镜和外泌体标志物进行鉴定。此外，通过共培养将这些外泌体重新引入NP-MSC细胞中，这通过荧光共聚焦显微镜得以证实。用外泌体miR-15a处理的NP-MSCs增加了聚集蛋白聚糖和胶原蛋白II的mRNA及蛋白水平，同时降低了ADAMTS4/5和MMP-3/-13的mRNA及蛋白水平。甲苯胺蓝染色证实，用外泌体miR-15a处理的NP-MSCs中软骨生成分化增加。用外泌体抗miR-15a处理的NP-MSCs抑制了聚集蛋白聚糖和胶原蛋白II的表达，同时增加了ADAMTS4/5和MMP-3/-13的表达，并降低了软骨生成分化。双荧光素酶报告基因检测显示，miR-15a直接靶向MMP-3并下调其表达。miR-15a的过表达增加了细胞增殖和集落形成，而与MMP3的联合过表达则抑制了miR-15a的作用。这在miR-15a存在时PI3K和Akt磷酸化的降低、Wnt3a和β-连环蛋白的上调中也很明显，但MMP3的过表达则显示出相反的效果。总体而言，这些数据表明，外泌体miR-15a通过PI3K/Akt和Wnt3a/β-连环蛋白轴下调MMP-3来促进NP-MSCs的软骨生成分化。
