Integrative circRNA landscape of intrauterine adhesions: putative ceRNA axes and circRNA-associated splicing usage...

Intrauterine adhesions (IUA) are fibrotic scars that impair endometrial regeneration, and compromise fertility. Emerging evidence implicates circular RNAs (circRNAs) in fibrotic remodeling, but it remains unclear how the circRNA landscape and circRNA-associated splicing programs coordinately link uterine contractility, endometrial cell-cycle control, and immune activation in IUA.

To address this question, we reanalyzed rRNA-depleted RNA sequencing data from IUA and controls (GSE224093) to assemble a high-confidence circRNA catalog using four independent circRNA callers, identify differential expression, and construct direction-consistent circRNA-miRNA-mRNA competing endogenous RNA (ceRNA) circuits. Gene set enrichment and CIBERSORT-based deconvolution were combined to relate circRNA modules to smooth muscle contractility, proliferative programs, and macrophage phenotypes. CircRNA-associated splicing (CAS) usage was quantified with SUVA to detect IUA-related shifts in back-splicing versus canonical splicing, and CAS-RNA-binding protein (RBP) co-expression networks were delineated using differentially expressed RBPs. Selected circRNAs, mRNAs, and RBPs were validated in an independent cohort of IUA and non-IUA endometrial samples by back-splice junction-spanning RT-qPCR and Western blotting.

We identified 1,724 high-confidence circRNAs arising from 1,143 host genes. ceRNA integration highlighted an upregulated circRNA, hsa-KDM4B_0007, converging on smooth muscle contractility genes MYH11 and PPP1R12B, and downregulated circRNAs hsa-LPAR3_0001 and hsa-PPFIA1_0013 converging on cell-cycle regulators CDC6 and CDCA5. Immune deconvolution indicated expansion of both M1 and M2 macrophages in IUA, and several DECs (e.g., hsa-PLOD2_0001 and hsa-FAM13B_0009) were tightly correlated with M1-enriched inflammatory signatures. CAS profiling uncovered widespread alterations in circRNA-associated splicing usage within pathways related to uterine contractility, EGFR signaling, and epithelial-mesenchymal transition. Integration with differentially expressed RBPs revealed a CAS-RBP network in which RBPs such as EXO1 and SORBS1 tracked with CAS events in EGFR adaptor genes (e.g., GAB1, PTK2) and other fibrogenesis-related regulators. Back-splice junction-spanning RT-qPCR confirmed the dysregulation of multiple DECs and their putative target mRNAs, and protein-level changes of selected RBPs were verified in clinical samples.

This study provides a mechanistic circRNA map that links ceRNA circuitry with CAS remodeling. The ceRNA and CAS-RBP modules we delineate generate testable hypotheses on circRNA-driven fibrotic remodeling and nominate circRNAs, CAS events, and RBPs as potential biomarkers and therapeutic entry points for IUA.