Molecular and Epigenetic Pathways Underlying Epithelial Damage and Repair in Necrotizing Enterocolitis via Multi-omics Approach

IntroductionNeonatal necrotizing enterocolitis (NEC) is a severe gastrointestinal disorder with high mortality, characterized by epithelial cell injury and compromised epithelial repair. The mechanisms underlying defective epithelial regeneration remain poorly understood despite advances in single-cell omics. Addressing these challenges is essential for elucidating the pathogenesis of NEC and identifying therapeutic targets to restore epithelial regeneration and replace the damaged epithelial layer. MethodsMulti-omics approaches were employed to investigate molecular and spatial changes in experimental NEC at epigenetic and transcriptomic levels. These included bulk RNA sequencing, single-nucleus RNA sequencing (snRNA-seq), single-nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq), and multiplexed error-robust fluorescence in situ hybridization (MERFISH) for spatial transcriptomics. Complementary in vitro experiments and in vivo mouse models were utilized to evaluate NEC phenotypes, intestinal tissue morphology, and organoid formation. ResultsChanges in cell type composition, transcriptional network remodeling, and chromatin accessibility were observed in the small intestine of neonatal mice with NEC. Chromatin accessibility significantly changed in epithelial cells, highlighting their pivotal roles in NEC. A marked reduction in intestinal stem cells (ISCs) and transit-amplifying cells, along with an increased proportion of enteroendocrine cells, indicates disrupted epithelial regeneration and functional differentiation. These changes correlated with disrupted WNT signaling and stem cell maintenance genes (e.g., Lgr5, Smoc2, Axin2) and activation of inflammatory and hypoxia-related pathways (e.g., Il6, Tnf). The epigenetic regulator Ezh2 was identified as a critical factor in maintaining LGR5+ ISCs and epithelial homeostasis. Knockdown of Ezh2 reduced stemness and proliferation-related gene expression and exacerbated inflammation. Reactivation of WNT signaling restored Ezh2 and Lgr5 expression, improving intestinal regeneration. ConclusionThis study reveals dynamic transcriptomic, epigenetic, and spatial changes in NEC and highlights Ezh2 as a key regulator of LGR5+ intestinal stem cell function and epithelial regeneration. These findings provide insights into NEC pathogenesis and a basis for therapies targeting Ezh2 and WNT signaling to restore intestinal integrity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/647851v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1ad89eaorg.highwire.dtl.DTLVardef@396282org.highwire.dtl.DTLVardef@172596forg.highwire.dtl.DTLVardef@4daf28_HPS_FORMAT_FIGEXP M_FIG C_FIG