CircGRB14 Inhibits Proliferation and Promotes Apoptosis of Granulosa Cells in Chicken Follicle Selection Through Sponging miR-12264-3p and miR-6660-3p

The development and selection of ovarian follicles are essential for continuous egg production in chickens. Non-coding RNAs, particularly circular RNAs (circRNAs), play a critical regulatory role in follicle selection, a process heavily involving granulosa cells (GCs). In this study, we analyzed circRNA expression profiles in small yellow follicles (SYFs) and large yellow follicles (LYFs) of Taihang chickens using RNA sequencing. We identified 14,586 circRNAs, with 57 showing differential expression (DE-circRNAs) between SYFs and LYFs. Functional enrichment analysis revealed that these DE-circRNAs are involved in key biological processes, including signal transduction, cell membrane formation, and nuclear enzymatic regulation. We focused on circGRB14, a circRNA derived from the growth factor receptor-bound protein 14 (GRB14) gene, as a potential regulator of follicle selection. Using qPCR, CCK-8 proliferation assays, and Annexin V/PI apoptosis analysis, we demonstrated that circGRB14 inhibits GC proliferation and promotes apoptosis. In contrast, miR-12264-3p and miR-6660-3p, validated as direct targets of circGRB14 via Dual-Luciferase Reporter assays, exhibited opposing effects by promoting GC proliferation and inhibiting apoptosis. These findings highlight the circGRB14-miR-12264-3p/miR-6660-3p axis as a key regulatory mechanism in GC dynamics during follicle selection. This study provides novel insights into the functional interplay between circRNAs and miRNAs in avian follicle development, offering potential targets for improving egg production in poultry.