piR‐23 Regulates Porcine Granulosa Cell Apoptosis by Targeting PTGS2

ABSTRACT Piwi‐interacting RNAs (piRNAs) are small noncoding RNAs that play roles in transposon regulation and gene silencing. Follicular atresia, a process involving granulosa cell (GC) apoptosis, is tightly linked to female reproductive efficiency. Previous studies suggested that piR‐23 is differentially expressed in porcine healthy (HF) and early atretic (AF) antral follicles, while prostaglandin‐endoperoxide synthase 2 (PTGS2), a key enzyme in prostaglandin biosynthesis, may be a potential target of piR‐23. This study investigated whether piR‐23 regulates GC apoptosis by targeting PTGS2. Porcine GCs were isolated from HF and AF. piR‐23 mimics/inhibitors and PTGS2 siRNA were transfected into GCs to assess cell apoptosis via Annexin V‐FITC/PI and CCK‐8 assays. Dual‐luciferase reporter assays validated the targeting of PTGS2 by piR‐23, while qRT‐PCR and Western blot analyzed PTGS2 expression. piR‐23 expression was downregulated in AF GCs. Overexpression of piR‐23 significantly reduced GC apoptosis, whereas inhibition of piR‐23 promoted apoptosis. PTGS2 expression was upregulated in AF GCs, and its knockdown suppressed GC apoptosis. Dual‐luciferase assays showed that piR‐23 directly bound to the 3′UTR of PTGS2, reducing its mRNA and protein levels. Cotransfection of piR‐23 inhibitor and PTGS2 siRNA reversed the proapoptotic effect of piR‐23 inhibition, confirming PTGS2 as a functional target. piR‐23 acts as an antiapoptotic regulator in porcine GCs by directly targeting PTGS2. This finding unveils a novel piRNA‐mediated regulatory mechanism in porcine follicular atresia.