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, little is currently known about whether miRNAs, an important class of epigenetic regulators, mediate the anti-atretic effect of TGF-β1. Here, a joint transcriptomic analysis demonstrated that miR-184, an anti-atretic miRNA, was significantly elevated by TGF-β1 in sow granulosa cells (GC). Quantitative detection, correlation analysis, luciferase reporter assay and ChIP confirmed that TGF-β1 induces miR-184 transcription in a SMAD4-dependent manner, while SMAD4 acts as a transcriptional activator to directly bind to miR-184 promoter under TGF-β1 stimulation. Interestingly, RNA-seq and bioinformatics analysis found that the targets 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 analysis revealed that miR-184, as a small-activating RNA (saRNA), activates TGF-β1 transcription by binding to its promoter and forming a RNA-induced transcriptional activation complex with AGO2, CTR9, DHX9, and RNA polymerase II. Therefore, a novel positive feedback loop, TGF-β1/SMAD4/miR-184, has been identified. In vitro GC and follicle culture systems were applied and validated that this loop exerts anti-apoptotic/atretic functions. 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 of 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.