Khor Yi-Sheng, Wong Pooi-Fong
Molecular Ageing Research Group, Department of Pharmacology, Faculty of Medicine, Universiti Malaya, 50603, Wilayah Persekutuan, Kuala Lumpur, Malaysia.
Biogerontology. 2025 Jul 21;26(4):147. doi: 10.1007/s10522-025-10290-3.
The accumulation of senescent endothelial cells within the endothelium leads to vascular dysfunction. Independent studies have linked miR-217, a senescence-associated microRNA and FOXO3, a longevity factor, to cellular senescence in different study models. However, their roles and direct interaction in endothelial replicative senescence (RS) remain to be investigated. This study sought to investigate the role of miR-217/FOXO3 axis in endothelial cell senescence. Young and RS HUVEC models were established to determine the differential mRNA and protein expressions of miR-217 and FOXO3 by RT-qPCR and immunoblotting. HUVECs were transfected with miR-217 mimic duplexes and hairpin inhibitors to evaluate the effects of elevated or reduced miR-217 expressions in endothelial health. FOXO3 was also stably knocked down using a lentiviral vector and rescue experiments were performed. RS HUVECs showed elevated senescence markers and miR-217 but decreased FOXO3 expressions. Dual-luciferase reporter assay confirmed FOXO3 as a direct miR-217 target. MiR-217 overexpression reduced FOXO3 but increased p16INK4a protein levels and impaired angiogenic potential in young HUVECs, suggesting that miR-217 promotes endothelial senescence and hampers angiogenesis by targeting FOXO3. Suppressing miR-217 increased FOXO3 expression in both RS and early-stage senescence (ES) HUVECs. However, ES but not RS HUVECs exhibited improved angiogenic potential. Meanwhile, FOXO3 knockdown increased miR-217 and p16INK4a expressions, indicating that FOXO3 loss drives endothelial senescence. However, transfection of FOXO3 knockdown cells with miR-217 inhibitor could only partially recover HUVECs angiogenic potential, suggesting that FOXO3 is critical for maintaining endothelial function. Collectively, these results showed that endothelial senescence and angiogenic potential are mediated through the crucial interaction of miR-217/FOXO3.
衰老内皮细胞在内皮中的积累会导致血管功能障碍。独立研究已将衰老相关的微小RNA miR-217和长寿因子FOXO3与不同研究模型中的细胞衰老联系起来。然而，它们在内皮细胞复制性衰老（RS）中的作用及直接相互作用仍有待研究。本研究旨在探究miR-217/FOXO3轴在内皮细胞衰老中的作用。建立了年轻和RS人脐静脉内皮细胞（HUVEC）模型，通过逆转录定量聚合酶链反应（RT-qPCR）和免疫印迹法确定miR-217和FOXO3的mRNA和蛋白质表达差异。用miR-217模拟双链体和发夹抑制剂转染HUVEC，以评估miR-217表达升高或降低对内皮健康的影响。还使用慢病毒载体稳定敲低FOXO3并进行了拯救实验。RS HUVEC显示衰老标志物和miR-217升高，但FOXO3表达降低。双荧光素酶报告基因检测证实FOXO3是miR-217的直接靶点。miR-217过表达降低了FOXO3，但增加了年轻HUVEC中p16INK4a蛋白水平并损害了血管生成潜力，表明miR-217通过靶向FOXO3促进内皮细胞衰老并阻碍血管生成。抑制miR-217可增加RS和早期衰老（ES）HUVEC中的FOXO3表达。然而，ES而非RS HUVEC表现出改善的血管生成潜力。同时，FOXO3敲低增加了miR-217和p16INK4a表达，表明FOXO3缺失驱动内皮细胞衰老。然而，用miR-217抑制剂转染FOXO3敲低细胞只能部分恢复HUVEC的血管生成潜力，表明FOXO3对维持内皮功能至关重要。总的来说，这些结果表明内皮细胞衰老和血管生成潜力是通过miR-217/FOXO3的关键相互作用介导的。
