Supplementary Material for: Bone Marrow Mesenchymal Stem Cells Release miR-378a-5p-Carried Extracellular Vesicles to Alleviate Rheumatoid Arthritis

This study investigates whether bone marrow mesenchymal stem cells (BMSCs)-derived extracellular vesicles (EVs) can affect rheumatoid arthritis (RA) by delivering microRNA (miR)-378a-5p to regulate the IRF1/STAT1 axis. We identified RA-associated miRNAs using GEO microarray dataset GSE121894. We found the most important miRNAs in RA synovial tissues using RT-qPCR. BMSCs-derived EVs were ultracentrifuged and co-cultured with HSMECs in vitro. Dual-luciferase and RIP studies examined miR-378a-5p's specific binding to IRF1. We also measured angiogenesis, migration, and proliferation using CCK-8, Transwell, and tube formation assays. CIA (collagen-induced arthritis) mice models were created by inducing arthritis and scoring it. RA synovial tissues had low miR-378a-5p expression, whereas BMSC-derived EVs had high levels. The transfer of miR-378a-5p by BMSC-derived EVs to HSMECs boosted proliferation, migration, and angiogenesis. miR-378a-5p inhibited IRF1. MiR-378a-5p-containing BMSC-derived EVs decreased STAT1 phosphorylation and HSMEC IRF1 expression. EVs with miR-378a-5p mimic promoted HSMEC proliferation, migration, and angiogenesis, whereas dexmedetomidine inhibited STAT1 phosphorylation. In CIA mice, BMSC-derived EVs containing miR-378a-5p enhanced synovial vascular remodeling and histopathology. Thus, miR-378a-5p from BMSC-derived EVs promotes HSMEC proliferation, migration, and angiogenesis, inactivating the IRF1/STAT1 axis and preventing rheumatoid arthritis.

本研究旨在探讨骨髓间充质干细胞（bone marrow mesenchymal stem cells, BMSCs）衍生的细胞外囊泡（extracellular vesicles, EVs）是否可通过递送微小RNA（microRNA, miR）-378a-5p调控IRF1/STAT1信号轴，进而影响类风湿关节炎（rheumatoid arthritis, RA）。研究人员借助GEO基因芯片数据集GSE121894筛选RA相关miRNA，并通过实时定量聚合酶链反应（RT-qPCR）明确RA滑膜组织中的核心miRNA。本研究采用超速离心法提取BMSCs来源的EVs，并将其与HSMECs进行体外共培养；通过双荧光素酶报告基因实验及RNA免疫沉淀（RIP）实验验证miR-378a-5p与IRF1的特异性结合。此外，本研究分别采用细胞计数试剂盒-8（CCK-8）、Transwell小室实验及管腔形成实验检测细胞的增殖、迁移与血管生成能力。通过诱导关节炎构建胶原诱导性关节炎（collagen-induced arthritis, CIA）小鼠模型，并对其进行关节炎评分。实验结果显示，RA滑膜组织中miR-378a-5p呈低表达，而BMSCs来源的EVs中该miRNA的表达水平较高；BMSCs来源的EVs将miR-378a-5p递送至HSMECs后，可促进细胞增殖、迁移及血管生成。miR-378a-5p可靶向抑制IRF1的表达；携带miR-378a-5p的BMSCs来源EVs可降低STAT1的磷酸化水平及HSMECs中IRF1的表达量。转染miR-378a-5p模拟物的EVs可促进HSMECs的增殖、迁移及血管生成，而右美托咪定可抑制STAT1的磷酸化。在CIA小鼠模型中，携带miR-378a-5p的BMSCs来源EVs可改善滑膜血管重构及组织病理学状态。综上，BMSCs来源的EVs携带的miR-378a-5p可通过抑制IRF1/STAT1信号轴的激活，促进HSMECs的增殖、迁移及血管生成，进而发挥类风湿关节炎的防治作用。