Supplementary Material for: Exosome miR-23a-3p From Osteoblast Alleviates Spinal Cord Ischemia/Reperfusion Injury by Down-regulating KLF3-activated CCNL2 Transcription

Background: Spinal cord ischemia/reperfusion injury (SCIRI) is usually caused by spinal surgery or aortic aneurysm surgery and can eventually lead to paralysis or paraplegia and neurological dysfunction. Exosomes are considered as one of the most promising therapeutic strategies for SCIRI as they can pass the blood-spinal barrier. Previous studies have proved that exosomes secreted by osteocytes have a certain slowing effect on SCIRI. Aim: We aimed to explore the effect of osteoblast secreted exosomes on SCIRI. Methods: Firstly, neurons and osteoblasts were co-cultured under different conditions. GEO database was utilized to detect the expression of miR-23a-3p in osteoblast exosomes. SCIRI cells were treated with exosomes, and the detection was taken to prove whether miR-23a-3p could slow the progression of SCIRI. Downstream gene and the potential regulatory mechanism were explored through database and functional experiments. Results: MiR-23a-3p was highly expressed in exosomes and it slowed down the process of SCIRI. Downstream mRNA KLF3 could bind to miR-23a-3p and was highly expressed in IRI. Moreover, CCNL2 was regulated by KLF3 and was highly expressed in IRI. Rescue experiments verified that miR-23a-3p suppressed the transcription of CCNL2 by targeting KLF3. Conclusion: Exosome miR-23a-3p from osteoblast alleviates SCIRI by down-regulating KLF3-activated CCNL2 transcription.

背景：脊髓缺血再灌注损伤（Spinal cord ischemia/reperfusion injury, SCIRI）通常由脊柱手术或主动脉瘤手术引发，最终可导致瘫痪、截瘫及神经功能障碍。外泌体（exosomes）因其可穿透血脊髓屏障，被认为是治疗SCIRI最具潜力的策略之一。既往研究证实，骨细胞分泌的外泌体对SCIRI具有一定的延缓作用。
目的：本研究旨在探讨成骨细胞（osteoblast）分泌的外泌体对SCIRI的影响。
方法：首先，在不同条件下对神经元与成骨细胞进行共培养。通过GEO数据库检测成骨细胞外泌体中miR-23a-3p的表达水平。将外泌体作用于SCIRI细胞模型，通过实验验证miR-23a-3p是否可延缓SCIRI的进展。借助数据库分析与功能实验，探究其下游靶基因及潜在调控机制。
结果：miR-23a-3p在外泌体中呈高表达，且可延缓SCIRI的进程。下游靶mRNA KLF3可与miR-23a-3p结合，且在缺血再灌注损伤模型中呈高表达。此外，CCNL2受KLF3调控，同样在缺血再灌注损伤模型中高表达。拯救实验证实，miR-23a-3p通过靶向KLF3抑制CCNL2的转录。
结论：成骨细胞来源的外泌体miR-23a-3p通过下调KLF3激活的CCNL2转录，从而缓解SCIRI。