FMR1 regulates immune-related gene expression and alternative splicing in human granulosa cells

Background The fragile X mental retardation 1 (FMR1) gene is strongly implicated in ovarian function, and premutation expansions of FMR1 represent the most common monogenic cause of premature ovarian insufficiency (POI). However, the ovarian-specific molecular mechanisms by which FMR1 dysregulation contributes to ovarian dysfunction remain poorly understood. In particular, the roles of FMR1 in coordinating gene expression and alternative splicing in granulosa cells have not been systematically explored. Methods Using lentiviral-mediated overexpression, we established stable FMR1-expressing KGN cell lines alongside wild-type controls, subsequently interrogating their transcriptomes through bulk RNA sequencing. Differential expressed genes were assessed using edgeR, followed by functional annotation using KOBAS 2.0 for Gene Ontology (GO) biological processes and KEGG pathway enrichment analyses. Alternative splicing events (ASEs) were identified with the ABLas pipeline. Selected genes and splicing events were validated by RT-qPCR. Results FMR1 overexpression induced substantial transcriptional reprogramming, with 3,008 differentially expressed genes identified—1,531 showing increased abundance, 1,477 decreased. Upregulated genes were predominantly enriched in type I interferon signaling and antiviral immune response pathways, whereas downregulated genes were mainly associated with cholesterol biosynthesis and steroid metabolic processes. RT-qPCR confirmed increased expression of BMP4, IRF7, IFI27, FAT1 , and PLAU , together with reduced expression of FABP3 and CSF1 (all P < 0.001). In parallel, FMR1 overexpression led to widespread alterations in alternative splicing across 4,077 genes, enriched for translation machinery, RNA processing, and splicing regulation. Intersection analysis identified 208 genes exhibiting coordinated changes in both expression levels and splicing patterns, with significant enrichment in innate immunity and cellular metabolism. RT-qPCR further confirmed significant splicing in four targets: IFI27 demonstrated reduced splice-form ratios (P < 0.001), while LAMC2 , IRF7 , and CSF1 exhibited elevated ratios (P < 0.05). Conclusion These findings reveal that FMR1 regulates immune-related and metabolic gene networks in human granulosa cells through coordinated modulation of gene expression and alternative splicing. This dual-layer regulatory effect provides mechanistic insight into how FMR1 dysregulation may contribute to ovarian dysfunction and FXPOI pathogenesis, and highlights alternative splicing as an important but previously underappreciated regulatory mechanism in ovarian biology.