Yang Yu, Shen Zhaoliang, Sun Wei, Gao Shuang, Li Yuanlong, Guo Yue
aDepartment of orthopaedics. The First Affiliated Hospital bJinzhou Medical University cJinzhou Second Hospital, Jinzhou, People's Republic of China.
Neuroreport. 2017 May 3;28(7):367-374. doi: 10.1097/WNR.0000000000000752.
To achieve neuronal differentiation of mouse bone mesenchymal stem cells (bMSCs) into neuron-like cells and explore the role of miR-122-5p that may regulate T-box brain 1 (Tbr1) expression during the induction. BMSCs were cultured and induced with butylated hydroxyanisole, retinoic acid (RA), basic fibroblast growth factor, and nerve growth factor in vitro. The cells were stained for neuron-specific enolase (NSE) and β-III-tubulin by immunocytochemistry/immunofluorescence. MiR-122-5p that may regulate Tbr1 expression was predicted by bioinformatics and identified using a Dual-Luciferase assay. The expressions of miR-122-5p and Tbr1 were determined by real-time PCR and western blot before and after the induction. After infection of miR-122-5p, the expressions of Tbr1, NSE, and tauons were measured. BMSCs showed a short spindle shape with a uniform distribution. After 14 days, the induced cells showed neuronal traits with a pyramidal appearance. TargetScan and miRanda showed that miR-122-5p was well complementary with the target site of the Tbr1 3'-untranslated region. Identified by the Dual-Luciferase assay, we found that miR-122-5p could inhibit Tbr1 expression by binding to its 3'-untranslated region. Furthermore, the expressions of Tbr1 mRNA and protein were decreased by real-time PCR and western blot. Overexpression of miR-122-5p downregulated the expressions of Tbr1, NSE, and tauons. MiR-122-5p may negatively regulate Tbr1 expression to affect the differentiation of bMSCs into neuron-like cells.
为实现小鼠骨髓间充质干细胞（bMSCs）向神经元样细胞的神经元分化，并探索在诱导过程中可能调节T-盒脑蛋白1（Tbr1）表达的miR-122-5p的作用。体外培养bMSCs并用丁基羟基茴香醚、视黄酸（RA）、碱性成纤维细胞生长因子和神经生长因子进行诱导。通过免疫细胞化学/免疫荧光对细胞进行神经元特异性烯醇化酶（NSE）和β-III-微管蛋白染色。通过生物信息学预测可能调节Tbr1表达的miR-122-5p，并使用双荧光素酶测定法进行鉴定。在诱导前后通过实时PCR和蛋白质印迹法测定miR-122-5p和Tbr1的表达。感染miR-122-5p后，测量Tbr1、NSE和微管相关蛋白的表达。bMSCs呈短梭形，分布均匀。14天后，诱导细胞呈现出具有金字塔形外观的神经元特征。TargetScan和miRanda显示miR-122-5p与Tbr1 3'-非翻译区的靶位点具有良好的互补性。通过双荧光素酶测定法鉴定，我们发现miR-122-5p可通过与其3'-非翻译区结合来抑制Tbr1表达。此外，实时PCR和蛋白质印迹法显示Tbr1 mRNA和蛋白质的表达降低。miR-122-5p的过表达下调了Tbr1、NSE和微管相关蛋白的表达。miR-122-5p可能通过负向调节Tbr1表达来影响bMSCs向神经元样细胞的分化。
