Combining network toxicology, single-cell RNA sequencing, and in vitro/in vivo experimental models reveals MMP9 as the key mediator of 27-hydroxycholesterol-driven ovarian cancer.

27-Hydroxycholesterol (27-HC), as a major cholesterol metabolite, has been shown to be associated with tumor biology, but its role in ovarian cancer remains unclear. This study integrates network toxicology, multi-cohort transcriptomic validation, and single-cell RNA sequencing to systematically investigate the potential role of 27-HC in ovarian cancer and identify key molecular mediators. Among 51 overlapping targets, MMP9 was identified as a robust candidate and consistently validated in independent datasets. Single-cell analysis revealed that MMP9 exhibited distinct expression patterns across different cell populations and was associated with extracellular matrix-related processes. Molecular docking and dynamic simulations confirmed a stable interaction between 27-HC and MMP9. Functionally, 27-HC significantly promoted ovarian cancer cell proliferation, cell cycle progression, and malignant phenotypes in vitro, and facilitated tumor growth in vivo. Concurrently, 27-HC treatment significantly upregulated MMP9 expression at both the mRNA and protein levels in ovarian cancer cells. In summary, these findings provide evidence supporting a potential role of 27-HC in ovarian cancer and its association with MMP9 upregulation, highlighting MMP9 as a potential mediator linking cholesterol metabolism to tumor progression.