Department of Orthopedics, Hunan Provincial People's Hospital, The First Affiliated Hospital of Hunan Normal University, Changsha 410000, China.
Acta Biochim Biophys Sin (Shanghai). 2018 Apr 1;50(4):345-354. doi: 10.1093/abbs/gmy017.
Dysfunction of cartilaginous endplates (CEP) is an important etiologic aspect of intervertebral disc degeneration (IDD) because the endplate has nutritional and biomechanical functions in maintaining proper disc health. In this study, we investigated the regulatory effects of estrogen on degenerated human CEP cells and the involvement of miR-221 in these effects. Normal and degenerated human CEP tissues were collected from patients with idiopathic scoliosis and IDD, respectively. CEP cells were isolated from these tissues. Polymerase chain reaction (PCR) and western blot analysis were performed to detect the expression of specific genes and proteins, respectively. Apoptosis and cell cycle were analyzed by flow cytometry. The results showed that the levels of aggrecan, collagen II, TGF-β and estrogen receptor α (ERα) were decreased in degenerated CEP tissues, while the levels of MMP-3, adamts-5, IL-1β, TNF-α, IL-6, and miR-221 were increased. Treatment of degenerated CEP cells with 17beta-estradiol (E2) increased the expressions of aggrecan and collagen II, as well as the secretion of TGF-β, but decreased IL-6 secretion. Moreover, E2 inhibited the apoptosis, resumed cell-cycle progression in G0/G1 phase, and improved the cell viability. These data indicate that estrogen has protective effect against degeneration of CEP cells. Furthermore, ERα was confirmed to be a target of miR-221 by the luciferase assay. The synthetic miR-221 mimics or knockdown of ERα attenuated the protective effects of E2, but miR-221 inhibitors promoted the protective effects of E2. These results suggest that miR-221 may impair the protective effect of estrogen in degenerated CEP cells through targeting ERα. This study reveals an important mechanism underlying the degeneration of CEP cells.
软骨终板（CEP）功能障碍是椎间盘退变（IDD）的一个重要病因学方面，因为终板在维持适当的椎间盘健康方面具有营养和生物力学功能。在这项研究中，我们研究了雌激素对退变人 CEP 细胞的调节作用以及 miR-221 在这些作用中的参与。分别从特发性脊柱侧凸和 IDD 患者中收集正常和退变的人 CEP 组织。从这些组织中分离 CEP 细胞。聚合酶链反应（PCR）和 Western blot 分析分别用于检测特定基因和蛋白质的表达。通过流式细胞术分析细胞凋亡和细胞周期。结果表明，退变 CEP 组织中聚集蛋白聚糖、胶原 II、TGF-β和雌激素受体 α（ERα）的水平降低，而 MMP-3、adamts-5、IL-1β、TNF-α、IL-6 和 miR-221 的水平升高。用 17β-雌二醇（E2）处理退变 CEP 细胞增加了聚集蛋白聚糖和胶原 II 的表达，以及 TGF-β的分泌，但减少了 IL-6 的分泌。此外，E2 抑制细胞凋亡，恢复 G0/G1 期细胞周期进程，并提高细胞活力。这些数据表明雌激素对 CEP 细胞退变具有保护作用。此外，通过荧光素酶测定证实 ERα是 miR-221 的靶标。合成 miR-221 模拟物或 ERα 的敲低减弱了 E2 的保护作用，但 miR-221 抑制剂促进了 E2 的保护作用。这些结果表明，miR-221 可能通过靶向 ERα 损害 E2 在退变 CEP 细胞中的保护作用。这项研究揭示了 CEP 细胞退变的一个重要机制。
