MiR-196a-5p/NR6A1 Regulates All-Trans Retinoic Acid Induced Neural Differentiation of Hntera-2 Stem Cells Via E-Cadherin Inhibition

Background: The differentiation of stem cells is a complex biological process requiring the involvement of multiple molecules and signaling pathways. Accumulating studies have reported that noncoding RNAs participate in the differentiation of stem cells and determination of cell fate. However, the role and regulation of miR-196a-5p in stem cell differentiation remain unclear. The aim of the study is to determine the regulatory mechanism of miR-196a-5p in stemness maintenance and directed differentiation.Methods: The hNtera-2 (NT-2) cell line, which was derived from embryonic carcinoma cells and has the characteristics of multipotent stem cells, was used as in vitro model for studying stem cell differentiation under the all-trans retinoic acid (RA) induction. The relationship and regulation between miR-196a-5p and NR6A1, NR6A1 and E-cadherin, were detected by RT-qPCR, western blot, MTT, Transwell, spheroid formation, immunofluorescence staining, Co-IP, CHIP and reporter assay.Findings: miR-196a-5p can inhibit the proliferation and metastasis as well as maintain the stemness of NT-2 cells through targeting the NR6A1 gene. During the RA-induced differentiation process, NR6A1 binds to the E-cadherin gene promoter region by recognizing the DR0 site and simultaneously recruits the methyltransferase Dnmt1 to inhibit the expression of E-cadherin and promote the switch of E-cadherin expression to N-cadherin expression. RNAi-mediated silencing of NR6A1 delayed the expression of neuron-specific genes during stem cell differentiation, which may lead to neurodevelopmental delay.Interpretation: These findings revealed that the miR-196a-5p/NR6A1/E-cadherin signaling axis is involved in the maintenance of NT-2 cell stemness and the inhibition of early neural differentiation.Funding Statement: The present study was supported by the National Natural Science Foundation of China (grant no. 81571494).Declaration of Interests: The authors have declared that no competing interest exists.