MiR-20b/106a modulate Ngn2 gene expression during neural differentiation of human umbilical cord mesenchymal stem cells.

Abstract

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.