Wei Guo-Jun, Zheng Ke-Wen, An Gang, Shi Zuo-Wei, Wang Kai-Fu, Guan Ying, Wang Yan-Song, Li Peng-Fei, Dong Da-Ming
Department of Orthopaedics, the 1st Affiliated Hospital of Harbin Medical University, Harbin, China.
Department of Orthopedics, Hongqi Hospital of Mudanjiang Medical University, Mudanjiang, China.
Cell Physiol Biochem. 2018;47(1):129-139. doi: 10.1159/000489756. Epub 2018 May 10.
BACKGROUND/AIMS: Transplantation of bone-marrow-derived mesenchymal stem cells (MSCs) promotes neural cell regeneration after spinal cord injury (SCI). Recently, we showed that suppression of microRNA-383 (miR-383) in MSCs increased the protein levels of glial cell line derived neurotrophic factor (GDNF), resulting in improved therapeutic effects on SCI. However, the overall effects of miR-383 suppression in MSCs on SCI therapy were not determined yet. Here, we addressed this question.
We used bioinformatics tools to predict all miR-383-targeting genes, confirmed the functional bindings in a dual luciferase reporter assay. The effects of alteration of candidate genes in MSCs on cell proliferation were analyzed by MTT assay and by Western blotting for PCNA. The effects on angiogenesis were assessed by HUVEC assay. The effects on SCI in vivo were analyzed by transplantation of the modified MSCs into nude rats that underwent SCI.
Suppression of miR-383 in MSCs not only upregulated GDNF protein, but also increased vascular endothelial growth factor A (VEGF-A) and cyclin-dependent kinase 19 (CDK19), two other miR-383 targets. MiR-383-suppression-induced increases in CDK19 resulted in a slight but significant increase in MSC proliferation, while miR-383-suppression-induced increases in VEGF-A resulted in a slight but significant increase in MSC-mediated angiogenesis.
Upregulation of CDK19 and VEGF-A by miR-383 suppression in MSCs further improve the therapeutic potential of MSCs in treating SCI in rats.
背景/目的：骨髓间充质干细胞（MSCs）移植可促进脊髓损伤（SCI）后神经细胞再生。最近，我们发现抑制MSCs中的微小RNA-383（miR-383）可增加胶质细胞源性神经营养因子（GDNF）的蛋白水平，从而改善对SCI的治疗效果。然而，MSCs中miR-383抑制对SCI治疗的总体效果尚未确定。在此，我们解决了这个问题。
我们使用生物信息学工具预测所有miR-383靶向基因，并在双荧光素酶报告基因检测中确认功能结合。通过MTT法和PCNA的蛋白质印迹分析MSCs中候选基因改变对细胞增殖的影响。通过人脐静脉内皮细胞（HUVEC）检测评估对血管生成的影响。通过将修饰后的MSCs移植到经历SCI的裸鼠体内分析对体内SCI的影响。
抑制MSCs中的miR-383不仅上调了GDNF蛋白，还增加了另外两个miR-383靶点血管内皮生长因子A（VEGF-A）和细胞周期蛋白依赖性激酶19（CDK19）。miR-383抑制诱导的CDK19增加导致MSCs增殖略有但显著增加，而miR-383抑制诱导的VEGF-A增加导致MSCs介导的血管生成略有但显著增加。
MSCs中miR-383抑制导致的CDK19和VEGF-A上调进一步提高了MSCs治疗大鼠SCI的潜力。
