Integrated Proteomics Reveals Spliceosome Dysregulation and Highlights DHX9 as a TGF‑β Signaling-Driven Fibrosis Hub in Endometriosis

Endometriosis (EM) is a chronic, estrogen-dependent disease characterized by the ectopic presence of endometrial-like tissue. Despite extensive research, its molecular pathogenesis remains unclear. In this study, we conducted an integrated proteomic network analysis using 22 endometrial samples to investigate protein-level alterations in ectopic lesions. Using weighted protein coexpression network analysis (WGCNA), we identified 12 protein modules strongly associated with EM clinical traits, with the turquoise module exhibiting significant downregulation of spliceosome-related proteins in ectopic tissues. Within this module, DHX9 emerged as a top hub protein, validated as significantly downregulated in ectopic (EC) tissues at both mRNA and protein levels. Functional studies demonstrated that DHX9 knockdown in endometrial stromal cells (ESCs) inhibited proliferation and, crucially, attenuated fibrogenesis. Proteomic profiling revealed that DHX9 knockdown downregulated proteins involved in the TGF-β signaling pathway and extracellular matrix organization. Consistent with this, DHX9 knockdown in a mouse endometriosis model significantly reduced ectopic lesion growth and collagen deposition. These findings establish DHX9 as a key regulator, promoting ESC fibrogenesis via TGF-β signaling, and implicate spliceosomal dysfunction in EM pathogenesis, highlighting DHX9 as a potential therapeutic target.