Abstract
Granulosa cells (GCs) are essential for follicular development, and their apoptosis is a key determinant of follicular atresia in mammals. Circular RNAs (circRNAs) have emerged as critical post-transcriptional regulators in reproductive biology; however, their roles in porcine GC apoptosis remain incompletely characterized. A novel circRNA derived from exon 2 of the INHBA gene (circINHBA) was identified from previous circRNA sequencing data. Its circular structure was verified by RNase R digestion assay, and subcellular localization was assessed by nucleocytoplasmic separation and fluorescence in situ hybridization. The functional role of circINHBA was investigated by siRNA-mediated knockdown, FACS, and Western blotting. The ceRNA mechanism was examined using dual-luciferase reporter assays, RNA pull-down, and RNA immunoprecipitation. circINHBA exhibited significantly higher expression in healthy follicles than in early atretic follicles and was predominantly localized in the cytoplasm. Knockdown of circINHBA promoted GC apoptosis and decreased the BCL2/BAX ratio. Mechanistically, circINHBA functioned as a molecular sponge for miR-196b-5p, which directly targets the 3'UTR of PARP8, thereby upregulating PARP8 expression to inhibit apoptosis. Furthermore, EIF4A3 physically interacted with circINHBA, and its knockdown significantly reduced circINHBA expression, indicating that EIF4A3 promotes circINHBA biogenesis. The EIF4A3/circINHBA/miR-196b-5p/PARP8 axis constitutes a novel regulatory mechanism governing GC apoptosis and follicular atresia in pigs, providing new insights into circRNA-RNA-binding protein crosstalk in ovarian physiology.