Nuclear factor IX promotes endometriosis progression through transcriptional activation of tetraspanin-2.

Endometriosis is a benign yet aggressive disease characterized by enhanced proliferation and invasion of ectopic endometrial tissue. Identifying upstream regulators that co-regulate these processes will provide novel insights into endometriosis pathogenesis and potential therapeutic targets. In this study, by integrating public single-cell RNA-seq data with our own RNA sequencing data, we identified nuclear factor IX (NFIX) as predominantly enriched in endometriotic stromal cells (ESCs), correlating with enhanced proliferative and invasive capacities. However, the underlying molecular mechanisms remain to be elucidated. Using the Venny platform, we intersected NFIX target genes from the KnockTF2.0 database with differentially expressed genes from our RNA sequencing data. Among these overlapping genes, we further identified tetraspanin-2 (TSPAN2) as a target of NFIX and validated that increased TSPAN2 expression mediated the regulatory effects of NFIX on ESCs' proliferation and invasion. Mechanistically, we found that NFIX exerts a significant stimulatory effect on TSPAN2 expression in ESCs. Luciferase reporter assays using serial deletion mutants confirmed that NFIX specifically binds to the -408 ~ -400 bp region of the TSPAN2 promoter, activating its transcription. Additionally, a chromatin immunoprecipitation (ChIP) assay revealed that the binding affinity of NFIX for the -408 ~ -400 bp region of the TSPAN2 promoter was higher in ESCs than in eutopic endometrial stromal cells (EMs). Moreover, NFIX knockdown in endometriosis mice downregulated TSPAN2 expression and inhibited ectopic lesion growth. Overall, this study demonstrated that NFIX promotes proliferation and invasion of ESCs by transcriptionally activating TSPAN2, suggesting NFIX as a potential therapeutic target. KEY MESSAGES: NFIX expression was markedly higher in ESCs than in EMs, correlating with increased proliferation and invasion capabilities. TSPAN2 was identified as a key mediator of NFIX-dependent regulation of proliferation and invasion of ESCs. NFIX transcriptionally activated TSPAN2 by binding to the -408 to -400 bp region of its promoter. In vivo knockdown of NFIX significantly inhibited the growth of endometriotic lesions in mice.