Wang Haoyu, Ban Wenrui, Wang Tao, Li Zhuo, Dang Xiaoqian
aThe Second Affiliated Hospital, Xi'an Jiaotong University, Xi'an bMedical Treatment College, Jinzhou Medical University cJinzhou Second Hospital, Jinzhou, People's Republic of China.
Neuroreport. 2017 Dec 13;28(18):1225-1231. doi: 10.1097/WNR.0000000000000900.
The aim of this study was to perform neural differentiation of mesenchymal stem cells derived from Wharton's jelly of human umbilical cord (HUMSCs) and explore the role of miR-20b and miR-106a, which may regulate Neurogenin-2 (Ngn2) expression during the neural differentiation. HUMSCs were cultured and induced in vitro. The cells were stained for nestin and microtubule-associated protein 2 (MAP2) by immunofluorescence. The interactional binding sites between the 3'-untranslated region (3'-UTR) of Ngn2 mRNA and miR-20b or miR-106a were predicted by bioinformatics and identified by a dual-luciferase assay. The expressions of Ngn2, miR-20b, and miR-106a were determined by real-time PCR and western blot before and after the neural differentiation. After infection of miR-20b or miR-106a, the expressions of Ngn2, MAP2, and β III-tubulin (TUBB3) were measured. HUMSCs showed a uniform pattern with a typical short spindle-shaped morphology. Fourteen days after neural differentiation, HUMSCs showed neuronal traits of pyramidal appearance. TargetScan and miRanda showed that miR-20b and miR-106a were well complementary with Ngn2 3'-UTR. Identified by the dual-luciferase assay, we found that miR-20b and miR-106a inhibit Ngn2 expression by binding to its 3'-UTR. Furthermore, the expression of Ngn2 mRNA was almost reciprocal to that of miR-20b and miR-106a by real-time PCR during the neural differentiation of HUMSCs. Overexpression of miR-20b and miR-106a downregulated the expressions of Ngn2, MAP2, and TUBB3. miR-20b and miR-106a may directly or indirectly regulate neuronal genes expression to modulate the neural differentiation of HUMSCs.
本研究的目的是对源自人脐带华通氏胶的间充质干细胞（HUMSCs）进行神经分化，并探索miR-20b和miR-106a的作用，它们可能在神经分化过程中调节神经生成素2（Ngn2）的表达。HUMSCs在体外进行培养和诱导。通过免疫荧光对细胞进行巢蛋白和微管相关蛋白2（MAP2）染色。通过生物信息学预测Ngn2 mRNA的3'-非翻译区（3'-UTR）与miR-20b或miR-106a之间的相互作用结合位点，并通过双荧光素酶测定进行鉴定。在神经分化前后，通过实时PCR和蛋白质印迹法测定Ngn2、miR-20b和miR-106a的表达。在感染miR-20b或miR-106a后，测量Ngn2、MAP2和β III-微管蛋白（TUBB3）的表达。HUMSCs呈现出均匀的模式，具有典型的短纺锤形形态。神经分化14天后，HUMSCs呈现出锥体外观的神经元特征。TargetScan和miRanda显示miR-20b和miR-106a与Ngn2 3'-UTR具有良好的互补性。通过双荧光素酶测定鉴定，我们发现miR-20b和miR-106a通过与其3'-UTR结合来抑制Ngn2的表达。此外，在HUMSCs的神经分化过程中，通过实时PCR检测到Ngn2 mRNA的表达与miR-20b和miR-106a的表达几乎呈相反关系。miR-20b和miR-106a可能直接或间接调节神经元基因的表达，从而调节HUMSCs的神经分化。
