Zhao Kunchi, Li Ran, Ruan Qing, Meng Chunyang, Yin Fei, Zhu Qingsan
Department of Spine Surgery, China-Japan Union Hospital of Jilin University, Changchun, China.
J Cell Mol Med. 2021 May 5;25(13):5924-39. doi: 10.1111/jcmm.16283.
The purpose of this study is to investigate the role of microRNA-125b (miR-125b) and its mechanism in spinal cord injury (SCI) by targeting Smurf1. After loss- and gain-function approaches were conducted in SCI rat models and neural stem cells (NSCs) isolated from foetal rats, the Basso-Beattie-Bresnahan (BBB) score was calculated, and related protein expression was determined by Western blot analysis and cell apoptosis by TUNEL staining. NSC viability was detected by CCK-8, migration abilities by Transwell assay and apoptosis by flow cytometry. The relationship between miR-125b, Smurf1 and KLF2 was evaluated by dual-luciferase reporter gene experiments, Co-IP and in vivo ubiquitin modification assays. Inhibition of miR-125b and KLF2 and the up-regulation of Smurf1 and ATF2 were observed in SCI rats. BBB scores were elevated, the expression of Nestin, NeuN, GFAP, NF-200 and Bcl-2 protein was enhanced but that of Bax protein was reduced, and cell apoptosis was inhibited in SCI rats after up-regulating miR-125b or silencing ATF2. Smurf1 was a target gene of miR-125b, which promoted KLF2 degradation through its E3 ubiquitin ligase function, and KLF2 repressed the expression of ATF2 in NSCs. The results in vivo were replicated in vitro. miR-125b overexpression promotes neurological function recovery after SCI.
本研究旨在通过靶向Smurf1来探讨微小RNA-125b（miR-125b）在脊髓损伤（SCI）中的作用及其机制。在对SCI大鼠模型以及从胎鼠分离的神经干细胞（NSCs）进行功能缺失和功能获得实验后，计算Basso-Beattie-Bresnahan（BBB）评分，并通过蛋白质免疫印迹分析测定相关蛋白表达，通过TUNEL染色检测细胞凋亡。采用CCK-8检测NSC活力，通过Transwell实验检测迁移能力，通过流式细胞术检测凋亡。通过双荧光素酶报告基因实验、免疫共沉淀和体内泛素化修饰实验评估miR-125b、Smurf1和KLF2之间的关系。在SCI大鼠中观察到miR-125b和KLF2受到抑制，Smurf1和ATF2上调。上调miR-125b或沉默ATF2后，SCI大鼠的BBB评分升高，巢蛋白、神经元核抗原、胶质纤维酸性蛋白、神经丝蛋白200和Bcl-2蛋白的表达增强，但Bax蛋白表达降低，细胞凋亡受到抑制。Smurf1是miR-125b的靶基因，其通过E3泛素连接酶功能促进KLF2降解，而KLF2抑制NSCs中ATF2的表达。体内实验结果在体外得到重复。miR-125b过表达促进SCI后神经功能恢复。
