Objective To investigate the gene expression profile in rat pancreatic ductal stem cells(PDSCs)when induced to differentiate into insulin-secreting cells(IPCs),with the goal of identifying key genes involved in this differentiation process.Methods The expanded PDSCs were categorized into a normal control(NC)group and an induced(Tre)group.PDSCs continued expansion culture in NC group,and cultured in induction medium for 28 days to facilitate the differentiation of PDSCs into IPCs in Tre group.Dithizone staining was employed to morphologically assess whether the cells exhibited a reddish-brown coloration,indicating a positive result.The immunofluorescence staining method was used to detect the expression of insulin(Ins)and PDX1 in the cells following induction.Additionally,ELISA was conducted to measure the Ins release from IPCs,thereby verifying the responsiveness of the induced cells to glucose-stimulated Ins secretion.Concurrently,cells were collected on induction days 0 and 28 for RNA sequencing(RNA-seq),and differentially expressed genes(DEGs)were analyzed and functionally annotated.The analysis revealed that regulatory factor X3(RFX3)was overexpressed in PDSCs,and the impact of RFX3 upregulation on differentiation induction was subsequently verified.Results Compared with NC group,DTZ staining was positive,PDX1 and Ins proteins were expressed,and an increased release of Ins in response to sugar stimulation was demonstrated in the Tre group.RNA-seq analysis identified 4270 DEGs,and functional enrichment analysis utilizing the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases revealed associations with Ins response,positive regulation of Ins secretion,pancreatic endocrine cell development,and overall pancreatic development.Additionally,functionally related genes such as ALDHA2,CREB5,EIF6,FOXO1,RFX3,WNT5a,OGT,GPR39,SMAD6,and TRPM2 were identified,indicating involvement in the cell cycle,TGF-β1 signaling pathway,FOXO signaling pathway,and Wnt signaling pathway in the regulation of the differentiation of pancreatic ductal stem cells(PDSCs)into insulin-producing cells(IPCs).Furthermore,the upregulation of RFX3 can inhibit the expression of TGF-β1 within 72 hours,thereby promoted the formation and release of Ins from insulin-positive cells.Conclusions Multiple genes and signaling pathways associated with pancreatic β-cell function collectively regulate the differentiation of rat PDSCs into IPCs.Notably,the upregulation of RFX3 enhances this differentiation process.

Objective To investigate the gene expression profile in rat pancreatic ductal stem cells(PDSCs)when induced to differentiate into insulin-secreting cells(IPCs),with the goal of identifying key genes involved in this differentiation process.Methods The expanded PDSCs were categorized into a normal control(NC)group and an induced(Tre)group.PDSCs continued expansion culture in NC group,and cultured in induction medium for 28 days to facilitate the differentiation of PDSCs into IPCs in Tre group.Dithizone staining was employed to morphologically assess whether the cells exhibited a reddish-brown coloration,indicating a positive result.The immunofluorescence staining method was used to detect the expression of insulin(Ins)and PDX1 in the cells following induction.Additionally,ELISA was conducted to measure the Ins release from IPCs,thereby verifying the responsiveness of the induced cells to glucose-stimulated Ins secretion.Concurrently,cells were collected on induction days 0 and 28 for RNA sequencing(RNA-seq),and differentially expressed genes(DEGs)were analyzed and functionally annotated.The analysis revealed that regulatory factor X3(RFX3)was overexpressed in PDSCs,and the impact of RFX3 upregulation on differentiation induction was subsequently verified.Results Compared with NC group,DTZ staining was positive,PDX1 and Ins proteins were expressed,and an increased release of Ins in response to sugar stimulation was demonstrated in the Tre group.RNA-seq analysis identified 4270 DEGs,and functional enrichment analysis utilizing the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases revealed associations with Ins response,positive regulation of Ins secretion,pancreatic endocrine cell development,and overall pancreatic development.Additionally,functionally related genes such as ALDHA2,CREB5,EIF6,FOXO1,RFX3,WNT5a,OGT,GPR39,SMAD6,and TRPM2 were identified,indicating involvement in the cell cycle,TGF-β1 signaling pathway,FOXO signaling pathway,and Wnt signaling pathway in the regulation of the differentiation of pancreatic ductal stem cells(PDSCs)into insulin-producing cells(IPCs).Furthermore,the upregulation of RFX3 can inhibit the expression of TGF-β1 within 72 hours,thereby promoted the formation and release of Ins from insulin-positive cells.Conclusions Multiple genes and signaling pathways associated with pancreatic β-cell function collectively regulate the differentiation of rat PDSCs into IPCs.Notably,the upregulation of RFX3 enhances this differentiation process.