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.