三氧化二砷通过circRNA-FOXO1-VEGF功能中心调控类风湿关节炎血管增殖的机制研究

Angiogenesis is critical for joint destruction in rheumatoid arthritis (RA), but the specific mechanism is unclear. Our previous study showed that RA fibroblast-like synoviocytes (RA-FLS) relevant angiogenic functional module in which VEGF played the key role was significantly up-regulated, and arsenic trioxide (ATO) could inhibit the synovial angiogenesis via this functional module. However, it is not clear how ATO achieves the specific location and regulation on the target genes. It has been reported recently that cyclic non-coding RNA (circRNA) can recruit transcription factors to the promoter of target genes. Our preliminary experiments showed that elevated circ-Raart in RA-FLS could bind to the transcription factor FOXO1 specifically, and furthermore ATO could inhibit the circ-Raart and FOXO1 expression particularly. Therefore, our hypothesis is that circ-Raart may recruit FOXO1 to up-regulate the expression of the target genes including VEGF; ATO may achieve its therapeutic effect on RA angiogenesis via inhibition of circRNA-FOXO1-VEGF functional module. In this project, we aim to perform gene transfection in vivo and in vitro, molecular interaction techniques such as RIP, FISH and confocal laser scanning microscopy, and so on. In order to elucidate the key function and mechanism of circ-Raart in RA pathogenesis, we also try to explore the new mechanism of ATO treatment to reduce its toxicity and improve its efficacy.

血管增殖是类风湿关节炎(RA)关节破坏的关键环节，具体机制不明。我们前期报道以VEGF为核心的RA成纤维样滑膜细胞（RA-FLS）血管增殖模块异常上调，三氧化二砷(ATO)可抑制此模块遏制血管新生来治疗RA，但ATO如何实现靶基因定位及调控尚未知。新近报道环状RNA(circRNA)可募集转录因子到靶基因启动子调控转录。预实验中，circ-Raart在RA-FLS显著上调，可特异性结合VEGF的转录因子FOXO1，ATO则特异性抑制circ-Raart及FOXO1表达。故我们推测circ-Raart募集FOXO1上调VEGF等靶基因表达，ATO可通过抑制circRNA-FOXO1-VEGF功能中心遏制RA血管新生。本项目拟采用体内外基因转染、RIP等分子互作、激光共聚焦FISH定位等技术，阐明circ-Raart在RA发病中的关键作用及机制，探索新的ATO治病机理，达到ATO减毒提效目的。

血管新生是类风湿关节炎（RA）发病机制中的关键事件，三氧化二砷（ATO）可通过抑制滑膜血管内皮生长因子（VEGF）为中心的功能模块治疗RA，但其确切机制尚不清。环状RNA（circRNA）在RA发病机制中的重要作用逐渐被关注，但其详细机制仍未知。本课题将RA成纤维样滑膜细胞（RA-FLS）与人真皮微血管内皮细胞（HDMECs）建立共培养体系，发现一种circRNA（circHIPK3）表达水平在TNF-α诱导的RA-FLS中显著增高，且共培养的RA-FLS及上清液中VEGF含量与circHIPK3表达水平一样显著升高，上清液诱导HDMECs迁移及小管形成能力显著提升。进一步研究表明circHIPK3可通过海绵吸附miR-149-5p靶向FOXO1抑制下游VEGF表达，形成血管生成调节模块；更为重要的是，ATO可通过靶向抑制该功能模块遏制血管生成治疗RA。在胶原诱导性关节炎（CIA）模型中，关节内注射腺相关病毒si-circHIPK3和/或ATO治疗进一步证实上述研究结果。我们阐明了circHIPK3在RA发病机制中的重要调控作用，发掘ATO治疗RA的新型保护性机制，提示ATO与circHIPK3沉默联合治疗的巨大潜力。此外，外泌体作为circRNA遗传信息的载体，被用作疾病治疗的新型药物递送载体，然而，滑膜间充质干细胞外泌体（SMSCs-Exo）负载的circRNA及其在RA进展中的作用尚不清楚。本研究发现SMSCs-Exo和circEDIL3过表达SMSCs-Exo（Ad-circEDIL3-SMSCs-Exo）均显著下调RA-FLS中VEGF表达及其诱导的血管新生，且Ad-circEDIL3-SMSCs-Exo作用更加显著，并发现circEDIL3/miR-485-3p/PIAS3 /STAT3/VEGF功能模块机制，为RA治疗提供了一种新型治疗方法。