iPSC-Derived Pancreatic Progenitors Lacking FOXA2 Reveal Alterations in miRNA Expression Targeting Key Pancreatic Genes

Abstract

Recently, we reported that forkhead box A2 (FOXA2) is required for the development of human pancreatic α- and β-cells. However, whether miRNAs play a role in regulating pancreatic genes during pancreatic development in the absence of FOXA2 expression is largely unknown. Here, we aimed to capture the dysregulated miRNAs and to identify their pancreatic-specific gene targets in pancreatic progenitors (PPs) derived from wild-type induced pluripotent stem cells (WT-iPSCs) and from iPSCs lacking FOXA2 (FOXA2–/–iPSCs). To identify differentially expressed miRNAs (DEmiRs), and genes (DEGs), two different FOXA2–/–iPSC lines were differentiated into PPs. FOXA2–/– PPs showed a significant reduction in the expression of the main PP transcription factors (TFs) in comparison to WT-PPs. RNA sequencing analysis demonstrated significant reduction in the mRNA expression of genes involved in the development and function of exocrine and endocrine pancreas. Furthermore, miRNA profiling identified 107 downregulated and 111 upregulated DEmiRs in FOXA2–/– PPs compared to WT-PPs. Target prediction analysis between DEmiRs and DEGs identified 92 upregulated miRNAs, predicted to target 1498 downregulated genes in FOXA2–/– PPs. Several important pancreatic TFs essential for pancreatic development were targeted by multiple DEmiRs. Selected DEmiRs and DEGs were further validated using RT-qPCR. Our findings revealed that FOXA2 expression is crucial for pancreatic development through regulating the expression of pancreatic endocrine and exocrine genes targeted by a set of miRNAs at the pancreatic progenitor stage. These data provide novel insights of the effect of FOXA2 deficiency on miRNA-mRNA regulatory networks controlling pancreatic development and differentiation. Graphical Abstract

Genome-wide differential expression profiling of long non-coding RNAs in FOXA2 knockout iPSC-derived pancreatic cells

“…Our recent study demonstrated that the absence of FOXA2 in iPSCs results in impaired differentiation into pancreatic islets, as evidenced by a notable decrease in the expression of pancreatic developmental genes [39]. Furthermore, we found that those downregulated genes are targets for several upregulated miRNAs in PPs lacking FOXA2 [40]. In this study, we employed the same iPSC model to examine the effect of FOXA2 depletion on the lncRNA pro le at pancreatic progenitor and pancreatic islet stages.…”

“…By analyzing RNA-seq results from PPs and pancreatic islets derived from WT-iPSCs and FOXA2 −/− iPSCs, we observed a decrease in the expression of critical pancreatic genes involved in the development and function of pancreatic islets, such as PDX1, NKX6.1, NEUROG3, NEUROD1, NKX2.2, INS, GCG, and others [40]. We conducted a network analysis combining these downregulated pancreatic genes with DE-lncRNAs.…”

“…Correlations between DE-lncRNAs and DE-mRNAs in iPSC-derived pancreatic progenitors Next, we sought to explore the potential function of the identi ed DE-lncRNAs in this study; therefore, we conducted a correlation analysis to link the DE-lncRNAs and their correlated DEGs, as previously reported [40] and from the online database of 305 pancreatic tissues (https://gtexportal.org/home/), which included a large number of genes associated with pancreatic islet development and function (Supplementary Table 3). Key downregulated pancreatic genes were selected for the network analysis, including PDX1, NKX6.1, FOXA2, RFX6, GATA6, GATA4, PTF1A, NEUROD1, NKX2.2, INSM1, FEV, DALL4, CPA2, ONECUT1, MNX1, GLIS3, PROX1, TCF7L2, HES6, NR5A2, PCSK1, HNF4G, CHGA, CHGB, GP2, and GCK (Fig.…”

“…The function of lncRNAs during pancreatic lineage speci cation is not fully understood. Our recent studies revealed that the expression of several genes involved in the development and function of pancreatic islet cells is dysregulated by FOXA2 de ciency [39,40]. Therefore, in the current study, we used established FOXA2 −/ − iPSC lines to investigate the effect of FOXA2 loss on the lncRNA pro les in the pancreatic progenitors (PP) and pancreatic islets derived from hiPSCs.…”

Genome-wide differential expression profiling of long non-coding RNAs in FOXA2 knockout iPSC-derived pancreatic cells

“…The function of lncRNAs during pancreatic lineage specification is not fully understood. Our recent studies revealed that the expression of several genes involved in the development and function of pancreatic islet cells is dysregulated by FOXA2 deficiency [ 3 , 39 ]. Therefore, in the current study, we used established FOXA2 −/ − iPSC lines to investigate the effect of FOXA2 loss on the lncRNA profiles in the pancreatic progenitors (PPs) and pancreatic islets derived from hiPSCs.…”