Abstract
BackgroundThe regulatory mechanism of insulin-producing cells (IPCs) differentiation from induced pluripotent stem cells (iPSCs) in vitro is very important in the phylogenetics of pancreatic islets, the molecular pathogenesis of diabetes, and the acquisition of high-quality pancreatic β-cells derived from stem cells for cell therapy.MethodsmiPSCs were induced for IPCs differentiation. miRNA microarray assays were performed by using total RNA from our iPCs-derived IPCs containing undifferentiated iPSCs and iPSCs-derived IPCSs at day 4, day 14, and day 21 during step 3 to screen the differentially expressed miRNAs (DEmiRNAs) related to IPCs differentiation, and putative target genes of DEmiRNAs were predicted by bioinformatics analysis. miR-690 was selected for further research, and MPCs were transfected by miR-690-agomir to confirm whether it was involved in the regulation of IPCs differentiation in iPSCs. Quantitative Real-Time PCR (qRT-PCR), Western blotting, and immunostaining assays were performed to examine the pancreatic function of IPCs at mRNA and protein level respectively. Flow cytometry and ELISA were performed to detect differentiation efficiency and insulin content and secretion from iPSCs-derived IPCs in response to stimulation at different concentration of glucose. The targeting of the 3′-untranslated region of Sox9 by miR-690 was examined by luciferase assay.ResultsWe found that miR-690 was expressed dynamically during IPCs differentiation according to the miRNA array results and that overexpression of miR-690 significantly impaired the maturation and insulinogenesis of IPCs derived from iPSCs both in vitro and in vivo. Bioinformatic prediction and mechanistic analysis revealed that miR-690 plays a pivotal role during the differentiation of IPCs by directly targeting the transcription factor sex-determining region Y (SRY)-box9. Furthermore, downstream experiments indicated that miR-690 is likely to act as an inactivated regulator of the Wnt signaling pathway in this process.ConclusionsWe discovered a previously unknown interaction between miR-690 and sox9 but also revealed a new regulatory signaling pathway of the miR-690/Sox9 axis during iPSCs-induced IPCs differentiation.
Highlights
The regulatory mechanism of insulin-producing cells (IPCs) differentiation from induced pluripotent stem cells in vitro is very important in the phylogenetics of pancreatic islets, the molecular pathogenesis of diabetes, and the acquisition of high-quality pancreatic β-cells derived from stem cells for cell therapy
We detected these two markers of mature β-cells in Induced pluripotent stem cell (iPSC)-derived Insulinproducing cell (IPC) on day 21 of step 3 to evaluate the efficiency of these insulin-secreting cells
No more insulin was induced when the glucose concentration increased to 30 mM, suggesting that these IPCs reached the upper limit of their insulin secretion capacity in response to glucose (Fig. 1e)
Summary
The regulatory mechanism of insulin-producing cells (IPCs) differentiation from induced pluripotent stem cells (iPSCs) in vitro is very important in the phylogenetics of pancreatic islets, the molecular pathogenesis of diabetes, and the acquisition of high-quality pancreatic β-cells derived from stem cells for cell therapy. Other miRNAs, such as miR-7 and miR-199b-5p, have been studied functionally and reported to selectively affect the development of pancreatic islets, promoting the proliferation of β-cells and miR-124a and regulating Foxa expression and intracellular signaling in β-cells [10,11,12]. These findings, as highlighted above, encouraged us to identify different layers of miRNA regulatory networks, which will provide greater insights into the roles of noncoding RNAs and help further elucidate β-cell biology, pancreas formation, and the molecular mechanisms of diabetes etiopathogenesis
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