Micro-RNA-124-5p promotes insulin producing cell differentiation through regulating transcriptional factor NKX6.1

Abstract

Aims: Differentiating human embryonic stem cells into pancreatic β cells has been proposed as a practical approach to managing diabetes. There have been several protocols attempting to generate β-like cells or insulin-producing cells (IPCs), but their low efficiency is a common issue. The expression level of Nkx6.1 is crucial for maintaining pancreatic β cell identity, while the proportion of PDX1 and Nkx6.1 double positive cells were not satisfied in the present protocols, leading to relative low efficiency in the differentiation into IPCs. This study aims to identify the mechanism underlying the regulation of Nkx6.1 during IPC differentiation and provide new insights for diabetes therapy. Methods: In the current study, human embryonic stem cell (hESC) line H1 was used to perform IPC specifications. Immunofluorescence, flow cytometry, and qPCR were conducted to analyze gene expression. In addition, insulin and C-peptide were measured through glucose-stimulated insulin secretion (GSIS) assays and ELISA. Results: We found that the transcription factor NKX6.1, a crucial inducer of early pancreatic development and IPC generation, was downregulated by micro-RNA-124-5p (miR-124-5p) in hESCs during IPC differentiation. Also, we observed that miR-124-5p was upregulated and bound to the 3’ untranslated region (3’ UTR) of NKX6.1 in pancreatic progenitor (PP), which subsequently suppressed PP differentiation. Moreover, inhibiting miR-124-5p induced the generation of IPCs. Conclusion: The current study results demonstrated an important role for miR-124-5p in regulating NKX6.1 expression, which appears to be a practical strategy for producing IPCs.

Keywords

• miR-124-5p
• Insulin-producing cells
• NKX6.1
• Human embryonic stem cells
• Diabetes