Human iPSC-based modeling reveals coding and non-coding transcriptomic alterations associated with RFX3 deficiency in pancreatic islets

Abstract Background RFX3 is a transcription factor (TF) critical for pancreatic endocrine development. Its loss impairs β-cell differentiation, promotes enterochromaffin cell (EC) generation, and induces apoptosis. However, the role of non-coding RNAs in mediating these effects remains poorly understood. Methods Using CRISPR/Cas9-derived RFX3 knockout (KO) human iPSC-derived pancreatic islets, we performed integrated transcriptomic analyses of coding and non-coding RNAs. Differentially expressed miRNAs (DEmiRs) and lncRNAs (DElncRNAs) were validated by RT-qPCR. Target prediction and competing endogenous RNA (ceRNA) network analyses were conducted to explore potential regulatory interactions affecting key endocrine genes. Results RFX3 deficiency induced widespread transcriptomic changes in human iPSC-derived pancreatic islets. Core β-cell markers and key pancreatic TFs were downregulated, alongside genes regulating ion channels, vesicle trafficking, metabolic sensing, and insulin secretion. Conversely, apoptotic and EC genes were upregulated. RFX3 KO islets exhibited significant alterations in miRNA profiles, including upregulation of miR-451a, miR-215-5p, miR-122-5p, miR-338-3p, miR-194-5p, miR-378a-3p , and the miR-29 family, which are predicted to target critical pancreatic endocrine genes. Several lncRNAs, including LINC00461, MIAT, RMST , and AC020916.1 , were downregulated, potentially influencing miRNA activity via ceRNA interactions. Integrated analyses identified core regulatory axes, such as miR-4455/INS, miR-122-5p/ARX, and miR-660-3p/GHRL, while apoptosis-related genes, including CASP, TNFSF10 , and TXNIP , were also predicted targets of dysregulated miRNAs. Conclusions Our findings reveal widespread alterations in non-coding RNA networks associated with RFX3 loss, highlighting a potential layer of post-transcriptional regulation linked to impaired pancreatic endocrine development. These results provide insights into how RFX3 deficiency may reshape islet transcriptomes, influencing β-cell maturation, function, and survival.