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<p>Sepsis-associated acute respiratory distress syndrome (SA-ARDS) is a severe disease with high morbidity and mortality. Emerging evidence indicates that ferroptosis plays a critical role in the development of SA-ARDS. However, the key regulatory targets and underlying molecular mechanisms remain to be further elucidated. Weighted gene co-expression network analysis (WGCNA) was performed based on the GSE66890 dataset to identify SA-ARDS-related gene modules. Candidate genes were screened by intersecting WGCNA module genes with ARDS-related targets from the GeneCards database and ferroptosis-related genes from FerrDb. An in vitro SA-ARDS model was established in HuLEC-5a cells using lipopolysaccharide (LPS). Cell viability, inflammatory cytokine levels, apoptosis, and ferroptosis-related parameters, including ROS, MDA, 4-HNE, and Fe<sup>2+</sup> levels, were evaluated using MTT, ELISA, flow cytometry, and corresponding assay kits. The mRNA and protein expression levels were determined by RT-qPCR and Western blot. The transcriptional regulation of topoisomerase II alpha (TOP2A) by GATA binding protein 2 (GATA2) was verified using JASPAR prediction, chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays. All cellular experiments were performed using three independent biological replicates. WGCNA identified the salmon and cyan modules as significantly associated with SA-ARDS. Intersection analysis identified TOP2A as a key ferroptosis-related gene in SA-ARDS. TOP2A was significantly upregulated in LPS-induced HuLEC-5a cells. Knockdown of TOP2A markedly alleviated LPS-induced cellular injury, as evidenced by enhanced cell viability, reduced levels of inflammatory cytokines (IL-6, IL-8, TNF-α), decreased apoptosis rate, and lowered ROS and Fe<sup>2+</sup> accumulation, reduced MDA and 4-HNE levels, restored GPX4 and SLC7A11 expression, and decreased ACSL4 expression, along with increased expression of endothelial barrier-related proteins. GATA2 directly bound to the TOP2A promoter and activated its transcription. TOP2A overexpression reversed the protective effects of GATA2 knockdown in LPS-induced HuLEC-5a cells. GATA2 transcriptionally activated TOP2A expression, which in turn promoted ferroptosis and exacerbated LPS-induced HuLEC-5a cell injury.</p> Graphical abstract <p></p>

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WGCNA-based identification and experimental validation of TOP2A as a key regulator of ferroptosis in sepsis-associated acute respiratory distress syndrome

  • 期刊论文

  • 发表日期：

    2026年7月24日

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  • Na Wang,
  • Kang Li,
  • Chuanyong Gong,
  • Jie Bi

摘要

Sepsis-associated acute respiratory distress syndrome (SA-ARDS) is a severe disease with high morbidity and mortality. Emerging evidence indicates that ferroptosis plays a critical role in the development of SA-ARDS. However, the key regulatory targets and underlying molecular mechanisms remain to be further elucidated. Weighted gene co-expression network analysis (WGCNA) was performed based on the GSE66890 dataset to identify SA-ARDS-related gene modules. Candidate genes were screened by intersecting WGCNA module genes with ARDS-related targets from the GeneCards database and ferroptosis-related genes from FerrDb. An in vitro SA-ARDS model was established in HuLEC-5a cells using lipopolysaccharide (LPS). Cell viability, inflammatory cytokine levels, apoptosis, and ferroptosis-related parameters, including ROS, MDA, 4-HNE, and Fe2+ levels, were evaluated using MTT, ELISA, flow cytometry, and corresponding assay kits. The mRNA and protein expression levels were determined by RT-qPCR and Western blot. The transcriptional regulation of topoisomerase II alpha (TOP2A) by GATA binding protein 2 (GATA2) was verified using JASPAR prediction, chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays. All cellular experiments were performed using three independent biological replicates. WGCNA identified the salmon and cyan modules as significantly associated with SA-ARDS. Intersection analysis identified TOP2A as a key ferroptosis-related gene in SA-ARDS. TOP2A was significantly upregulated in LPS-induced HuLEC-5a cells. Knockdown of TOP2A markedly alleviated LPS-induced cellular injury, as evidenced by enhanced cell viability, reduced levels of inflammatory cytokines (IL-6, IL-8, TNF-α), decreased apoptosis rate, and lowered ROS and Fe2+ accumulation, reduced MDA and 4-HNE levels, restored GPX4 and SLC7A11 expression, and decreased ACSL4 expression, along with increased expression of endothelial barrier-related proteins. GATA2 directly bound to the TOP2A promoter and activated its transcription. TOP2A overexpression reversed the protective effects of GATA2 knockdown in LPS-induced HuLEC-5a cells. GATA2 transcriptionally activated TOP2A expression, which in turn promoted ferroptosis and exacerbated LPS-induced HuLEC-5a cell injury.

Graphical abstract