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Identification of key mitochondria-related genes in necrotizing enterocolitis using single-cell hdWGCNA and experimental verification

Pediatric Research (2026)Cite this article

Abstract

Background

Necrotizing enterocolitis (NEC) is a severe gastrointestinal disorder in preterm infants. The interplay between mitochondrial metabolism and immune inflammation in its development is not fully understood.

Methods

Single-cell data were analyzed using dimensionality reduction, clustering, and the high-dimensional weighted gene co-expression network analysis (hdWGCNA) algorithm to identify key gene modules in monocytes. GSE46619 was integrated with the MitoCarta3.0 to identify mitochondria-associated differentially expressed genes (MitoDEGs). Acyl-CoA synthetase long-chain family member 1 (ACSL1) was selected as a candidate. Immune infiltration was evaluated via the CIBERSORT algorithm, and a competing endogenous RNA (ceRNA) regulatory network was constructed using Cytoscape. The expression and function of ACSL1 were validated both in vivo and in vitro, using immunohistochemistry (IHC), qRT-PCR, western blot, and siRNA knockdown.

Results

A key monocyte subset was identified in NEC. Integrated analysis revealed three MitoDEGs (ACSL1, SOD2, SLC25A37) were linked to NEC, with ACSL1 showing the most significant upregulation. ACSL1 expression correlated strongly with immune cell infiltration and was confirmed to be elevated in vivo and in vitro models. Knocking down ACSL1 suppressed lipopolysaccharide (LPS)-induced inflammatory factor expression and ROS production.

Conclusion

ACSL1 plays a critical role in the pathogenesis of NEC, suggesting its potential as a novel biomarker.

Impact

  • Our study reveals massive monocyte infiltration and identifies the mitochondria-related gene ACSL1 as highly expressed and functionally significant in NEC.

  • This is the first integrated analysis (single-cell, hdWGCNA, MitoCarta3.0) pinpointing ACSL1 as a novel immunometabolic hub specific to NEC pathophysiology.

  • ACSL1 provides a crucial mechanistic link between mitochondrial function and NEC development.

  • It significantly correlates with key immune cells (neutrophils/mast cells/macrophages/T cells), highlighting its role in NEC immune dysregulation.

  • These findings reveal a critical role of ACSL1 in NEC pathogenesis, highlighting its potential as a novel biomarker.

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Fig. 1: Single-cell analyses identify monocyte subpopulations as key cellular components in NEC.
Fig. 2: Central modules identified by hdWGCNA that are closely associated with monocytes in NEC.
Fig. 3: Pseudo-time trajectory analysis.
Fig. 4: Identification of DEGs in NEC and correlation analysis of ACSL1.
Fig. 5: Establishment of an animal model of NEC and confirmation of ACSL1 expression in NEC mice.
Fig. 6: Elevation of ACSL1 Expression in NEC and Inhibition of Inflammatory Response and ROS by ACSL1 Knockdown.

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Data availability

All data generated for this manuscript have been included in this article. The datasets generated and analyzed during this study are available from the corresponding author upon reasonable request.

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Author information

Author notes

  1. These authors contributed equally: Yuehua Chen, Kexin Gao.

Authors and Affiliations

  1. Department of Pediatric Surgery, Children’s Hospital of Soochow University, Suzhou, Jiangsu, China

    Yuehua Chen & Jian Wang

  2. Department of Pediatric Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China

    Yuehua Chen, Kexin Gao, Ning Chen & Qiyou Yin

Authors
  1. Yuehua Chen
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  2. Kexin Gao
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  3. Ning Chen
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  4. Qiyou Yin
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  5. Jian Wang
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Contributions

All the authors made substantial contributions to the study. J.W. and Q.Y. contributed to the study conception and design. Y.C. and K.G. conducted the experiments, collected and analyzed the data. N.C. wrote the original draft of the manuscript and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Qiyou Yin or Jian Wang.

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Chen, Y., Gao, K., Chen, N. et al. Identification of key mitochondria-related genes in necrotizing enterocolitis using single-cell hdWGCNA and experimental verification. Pediatr Res (2026). https://doi.org/10.1038/s41390-026-04779-x

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