MiR-20b/106a Modulate Neural Differentiation Of Human Umbilical Cord Mesenchymal Stem Cells By Targeting Ngn2 Expression

ObjectiveInvestigate the regulatory effect of miRNA on the differentiation of HUMSCs and its relationship with Ngn2.Methods1,Target Scan and miRanda were used to predict and to get the intersection,miR-20 b and miR-106 a.Dual-Luciferase assay was employed to verify miR-20 b or miR-106 a the regulatory effect on Ngn2 expression.2,We isolated HUMSCs from human umbilical cords and cultured in vitro and induced neural differentiation of HUMSCs with a modified two-step protocol.The expressions of two neuronal genes MAP2 and nestin were detected by immunofluorescence staining.Real-time PCR was employed to measure Ngn2 m RNA and miRNAs expression levels.3,HUMSCs were transfected with miR-20 b and miR-106 a using lentivirus.Expressions of the neuronal gene Ngn2,MAP2 and TUBB3 were detected by real-time PCR and western blot.Results1,Mi Randa displayed two miRNAs had only one target site on Ngn2 m RNA3’UTR Target Scan showed the target site of two miRNAs was the same and both miRNA families were broadly conserved among vertebrates.Dual-Luciferase assay showed miR-20 b and miR-106 a could inhibit Ngn2 expression by binding to its 3′UTR.2,Immunofluorescence staining revealed that MAP2 and nestin expressed in HUMSCs after inducing 14 days.After 7days Ngn2 m RNA expression was rapidly risen and increased to the maximum after 14 days.Instead,the expression of miR-20 b and miR-106 a after 7 days was rapidly decreased and declined to the minimum after 14 days.3,After over-expression of miR-20 b and miR-106 a,the expression levels of miR-20 b and miR-106 a were significantly increased at 48 h after transfection of lentiviral vectors and Ngn2 expression levels of m RNA and protein were significantly decreased in miR-20 b and miR-106a-transfected groups.The m RNA and protein levels of MAP2 and TUBB3 were also significantly declined in miR-20 b and miR-106a-transfected groups.ConclusionsmiR-20 b and miR-106 a may directly or indirectly regulate neuronal genes expression to modulate neural differentiation of HUMSCs and Ngn2-miR-20b/106 a autoregulatory feedback loop involved in the neural differentiation process.