Deciphering NEUROD1's target genes and miRNAs: an integrative analysis in small cell lung cancer

Small cell lung cancer (SCLC) is a recalcitrant tumor with a dismal prognosis. ASCL1 and NEUROD1 are master regulators defining the molecular subtypes of SCLC with neuroendocrine features. Immunohistochemical studies have shown that these genes can be co-expressed to varying degrees. While the transcriptional functions of ASCL1 are well-documented, those of NEUROD1, specifically its target genes and miRNAs, remain less explored. In this study, we utilized CRISPR-Cas9 to knockout NEUROD1 in a SCLC cell line expressing both ASCL1 and NEUROD1. RNA and microRNA sequencing analyses, integrated with genome-wide profiling of chromatin modifications by the CUT&Tag assay, revealed that NEUROD1 regulates a distinct subset of genes and miRNAs, including <italic>NEUROD2</italic>, <italic>NHLH2</italic>, and miR-139-5p. NEUROD1 was found to be strongly associated with NEUROD and NHLH transcription factors, potentially shaping molecular features distinct from the ASCL1-positive SCLC subtype. Furthermore, we discovered that miR-139-5p and its host gene <italic>PDE2A</italic> characterize the features of the NEUROD1-positive SCLC subtype. Administration of mimic miRNA demonstrated that miR-139-5p regulates specific genes. The interaction of miRNAs, particularly miR-139-5p, appears to enhance the characteristics of the NEUROD1-positive SCLC subtype. Our integrative analyses reveal novel aspects of NEUROD1-mediated transcriptomic and epigenetic regulation, contributing to the cellular heterogeneity of SCLC.