ceRNA regulatory networks reveals gga-miR-30b-5p as a regulator of granulosa cell apoptosis via targeting NR5A2 in atrophic ovaries of broody chickens

Reproductive efficiency forms the cornerstone of poultry production, yet ovarian oxidative stress and brooding that emerge with advancing age accelerate follicular atresia, leading to ovarian atrophy. This subsequently disrupts ovulation and shortens the laying cycle. Despite its economic implications, the molecular mechanisms linking these processes to granulosa cell (GC) fate remain poorly understood. This study aimed to construct ceRNA regulatory networks and unravel how microRNA-30b-5p (miR-30b-5p) and its target gene nuclear receptor subfamily 5 group A member 2 (NR5A2) regulate GC proliferation and apoptosis in chicken atrophic ovaries. In this study, we found that the ovarian tissue undergoes degeneration, with reduced levels of the hormones follicle-stimulating hormone (FSH) and luteinizing hormone (LH), and a significant decrease in ovarian antioxidant activity of broody chickens. Whole-transcriptome sequencing on atrophic ovaries (AO) from 700-day-old Hy-line Gray broody hens and normal ovaries (NO) from actively laying hens identified 260 differentially expressed (DE) lncRNAs, 492 DE circRNAs, 53 DE miRNAs, and 4,493 DE mRNAs. Further, integrative analysis revealed miR-30b-5p as a candidate regulator of GC proliferation and apoptosis. Additionally, functional assays including RT-qPCR, western blotting, flow cytometry, cell counting kit 8 (CCK-8) and 5-ethynyl-20-deoxyuridine (EdU) assay incorporation demonstrated that miR-30b-5p overexpression suppressed GC proliferation (reduced CDK2, Cyclin D1, PCNA, and Bcl-2) and promoted apoptosis (increased Caspase-3 and Caspase-9). Thereafter, target prediction and dual-luciferase reporter assays further confirmed that NR5A2 as a direct target of miR-30b-5p, and that NR5A2 knockdown phenocopied the anti-proliferative and pro-apoptotic effects of miR-30b-5p. Taken together, the findings obtained from this study demonstrate that miR-30b-5p promotes GC apoptosis and inhibits proliferation by targeting NR5A2, thereby contributing to follicular atresia during broodiness in the late laying phase. Targeted modulating of miR-30b-5p may offer a promising strategy to alleviate ovarian atrophy, extend the laying cycle and enhance economic efficiency in poultry production.