Table5_Potential predictive and therapeutic applications of small extracellular vesicles-derived circPARD3B in osteoarthritis.XLSX

Background: Heterogeneous phenotypes that display distinct common characteristics of osteoarthritis (OA) are not well defined and will be helpful in identifying more customized therapeutic options for OA. Circular RNAs (circRNAs) have attracted more and more attention due to their role in the progression of OA. Investigating the role of circRNAs in the pathogenesis of OA will contribute to the phenotyping of OA and to individualized treatment.
Methods: Small extracellular vesicles (sEV) were isolated from serum samples from patients with OA of different stages and sEV-derived circPARD3B was determined using RT-qPCR analysis. CircPARD3B expression in a stimulated coculture that included OA fibroblast-like synoviocytes (OA-FLS) as well as human dermal microvascular endothelial cells (HDMECs), plus the effects of circPARD3B on the expression of vascular endothelial growth factor (VEGF) long with angiogenic activity, were evaluated in vitro. Based on bioinformatics analysis and luciferase reporter assay (LRA), MiR-326 and sirtuin 1 (SIRT1) were found to be interactive partners of circPARD3B. Mesenchymal stem cells (SMSCs) overexpressing circPARD3B were constructed and SMSCs-derived sEV with overexpressed circPARD3B (OE-circPARD3B-SMSCs-sEV) were obtained to explore the effect of the intervention of circPARD3B combined with SMSCs-sEV-based therapy in vitro and in a OA model induced by collagenase in vivo.
Results: Serum sEV-linked circPARD3B was indentified to be significantly decreased in the inflammatory phenotype of OA. Overexpression of circPARD3B was found to inhibit the expression of VEGF, as well as the angiogenesis induced by VEGF in a IL-1β stimulated the co-culture of OA-FLS as well as HDMECs. CircPARD3B is directly bound to miR-326. SIRT1 was considered a novel miR-326 target gene. OE-circPARD3B-SMSCs-sEV significantly reduced VEGF expression in coculture of OA-FLS and HDMECs. Injection of OE-circPARD3B-SMSCs-sEV could also reduce synovial VEGF; additionally, it could further ameliorate OA in the mouse model of OA in vivo.
Conclusion: Serum sEV circPARD3B is a potential biomarker that enables the identification of the inflammatory phenotype of patients with OA. Correspondingly, intracellular transfer of circPARD3B through OE-circPARD3B-SMSCs-sEV could postpone disease progression through a functional module regulated angiogenesis of circPARD3B-miR-326-SIRT1, providing a novel therapeutic strategy for OA.

研究背景：表现出骨关节炎（osteoarthritis, OA）特有共性特征的异质性表型目前尚未得到明确界定，而明确此类表型有助于为骨关节炎患者开发更多定制化治疗方案。环状RNA（circular RNAs, circRNAs）因其在骨关节炎进展中的调控作用而受到越来越多的关注。探究circRNAs在骨关节炎发病机制中的功能，将有助于明确骨关节炎表型并推动个体化治疗的发展。
研究方法：从不同分期骨关节炎患者的血清样本中分离小细胞外囊泡（small extracellular vesicles, sEV），并通过实时定量聚合酶链式反应（RT-qPCR）检测sEV来源的circPARD3B的表达水平。在包含骨关节炎成纤维样滑膜细胞（OA fibroblast-like synoviocytes, OA-FLS）与人类皮肤微血管内皮细胞（human dermal microvascular endothelial cells, HDMECs）的刺激共培养体系中，检测circPARD3B的表达情况，并体外评估circPARD3B对血管内皮生长因子（vascular endothelial growth factor, VEGF）表达及血管生成活性的影响。通过生物信息学分析与荧光素酶报告基因实验（luciferase reporter assay, LRA），证实miR-326与沉默信息调节因子1（sirtuin 1, SIRT1）是circPARD3B的相互作用靶点。构建过表达circPARD3B的间充质干细胞（mesenchymal stem cells, SMSCs），并提取其分泌的过表达circPARD3B的sEV（OE-circPARD3B-SMSCs-sEV），以此探究circPARD3B干预联合SMSCs-sEV疗法在体外及胶原酶诱导的骨关节炎小鼠体内模型中的治疗效果。
研究结果：在骨关节炎炎症表型患者的血清sEV中，circPARD3B的表达水平显著降低。在经IL-1β刺激的OA-FLS与HDMECs共培养体系中，过表达circPARD3B可抑制VEGF的表达以及VEGF诱导的血管生成。circPARD3B可直接结合miR-326，而SIRT1是miR-326的新型靶基因。OE-circPARD3B-SMSCs-sEV可显著降低OA-FLS与HDMECs共培养体系中的VEGF表达水平。体内注射OE-circPARD3B-SMSCs-sEV还可降低小鼠关节滑膜组织中的VEGF表达，进一步改善胶原酶诱导的骨关节炎小鼠模型的病症。
研究结论：血清sEV来源的circPARD3B可作为识别骨关节炎炎症表型患者的潜在生物标志物。相应地，通过OE-circPARD3B-SMSCs-sEV实现circPARD3B的细胞内递送，可通过circPARD3B-miR-326-SIRT1调控的血管生成功能模块延缓疾病进展，为骨关节炎提供了全新的治疗策略。