A novel positive feedback regulatory loop, TGF-β1/SMAD4/miR-184, controls the follicular development in sows

TGF-β1, the core ligand of the transforming growth factor β (TGF-β) signaling pathway, is crucial for follicular development and female fertility. However, it remains unclear whether microRNAs (miRNAs), an important class of epigenetic regulators, mediate the anti-atretic effect of TGF-β1. Here, joint transcriptomic analysis demonstrated that miR-184, an anti-atretic miRNA, was significantly elevated by TGF-β1 in sow granulosa cells (GCs). Quantitative detection, correlation analysis, luciferase reporter assays, and chromatin immunoprecipitation confirmed that TGF-β1 induces miR-184 transcription in a SMAD4-dependent manner, with SMAD4 acting as a transcriptional activator that directly binds to the miR-184 promoter under TGF-β1 stimulation. RNA-seq and bioinformatics analysis revealed that the target genes of miR-184 are enriched in the TGF-β pathway, with TGF-β1 being feedback-induced by miR-184. In vivo and in vitro mechanistic analyses revealed that miR-184, a small-activating RNA (saRNA), activates TGF-β1 transcription by binding to its promoter and forming an RNA-induced transcriptional activation complex with AGO2, CTR9, DHX9, and RNA polymerase II. Consequently, a novel positive feedback loop (TGF-β1/SMAD4/miR-184) was identified. In vitro GC and follicle culture systems validated the anti-apoptotic/atretic functions of this loop. Moreover, comparative analyses showed that the levels of TGF-β1 and miR-184 in the follicles of high-fertility sows were significantly higher than those in low-fertility sows. Our findings highlight the critical role of the interaction between TGF-β1 and miR-184 in inhibiting GC apoptosis and follicular atresia, providing potential targets for improving follicular development and sow fertility.