Multi-omics analysis reveals RBPJ-mediated regulation of EGF/ACTN2/MYPN/COL21A1 in fibroblast during oviduct...

The recombinant signal binding protein for immunoglobulin kappa J region (RBPJ) is the central effector of the Notch signaling pathway, which is a highly conserved pathway that regulates developmental and differentiation processes impacting tissue development, morphology, and function. The duck oviduct undergoes dramatic morphological and functional remodeling as laying egg or not, wherein fibroblasts play critical roles in extracellular matrix (ECM) remodeling and tissue homeostasis. However, the regulatory role of RBPJ in duck oviduct fibroblasts remains poorly understood. This study investigated the effects of RBPJ on the proliferation and molecular networks of duck fibroblasts using a RBPJ-overexpression model combined with RNA-seq and LC-MS/MS-based proteomic analysis. A RBPJ-overexpressing fibroblast model was successfully established. Compared to the control group (C), RBPJ overexpression (OE) significantly increased cell proliferation at 24 h (P < 0.01), as verified by CCK-8 and EdU assays. RNA-seq identified 130 differentially expressed genes (DEGs; |log2(fold change) | ≥ 1, P < 0.05), with significant enrichment of the Cytoskeleton in muscle cells pathway. Proteomic analysis detected 569 differentially expressed proteins (DEPs; |Fold change| ≥1.5, P < 0.05), primarily enriched in ECM-receptor interaction. Integrated transcriptome-proteome analysis revealed 12 key molecules. Among which, EGF is well-documented regulators of cell proliferation; ACTN2 (Actinin alpha 2), MYPN (Myopalladin) and COL21A1 (Collagen type XXI alpha 6 chain) were significantly enriched in pathways including ECM-receptor interaction, Cytoskeleton in muscle cells, and Focal adhesion. Collectively, these results indicate that RBPJ promotes fibroblast proliferation and regulates structural modulating by EGF, ACTN2, MYPN and COL21A1 at both the transcriptional and protein levels. Tissue-level verification demonstrated that the mRNA and protein expression levels of RBPJ in the oviduct of laying ducks were significantly elevated compared to ceased laying ducks (P < 0.05). Furthermore, the expression patterns of the 12 key DEGs in tissues aligned with the findings from cell study. This study provides novel molecular insights into RBPJ-mediated regulation of oviduct fibroblast function, offering a better understanding of duck oviduct remodeling during the laying cycle.