MiR-4670-3p promotes sheep granulosa cell proliferation through RORA/TGF-β/SMAD signaling pathway

Retinoic Acid Receptor-Related Orphan Receptor Alpha (RORA) can affect lambing numbers and cell proliferation in sheep. MicroRNAs (MiRNAs) are important for post-transcriptional gene regulation, but how they function during follicular development is currently unknown. In this study, miR-4670-3p was identified as a key microRNA targeting RORA using bioinformatics analysis. In ovarian tissues from sheep with high-fertility, miR-4670-3p abundance was substantially increased in comparison to the low-fertility group, demonstrating an inverse association with RORA levels. To validate this interaction, dual luciferase assay showed that miR-4670-3p suppressed RORA expression. To explore the functional roles of miR-4670-3p and RORA during development and growth of sheep granulosa cells (GCs), we generated specific overexpression and interference (inhibition) plasmids targeting each molecule. Enhanced expression of miR-4670-3p markedly increased the rate of GC proliferation, whereas inhibition of miR-4670-3p repressed GC proliferation. RORA overexpression or inhibition exhibited opposite effects to those mediated by miR-4670-3p on GC proliferation. Moreover, RORA overexpression also suppressed the expression of TGF-β/SMAD signaling pathway marker genes, whereas RORA inhibition produced the opposite effect. Cotransfection experiments with miR-4670-3p and RORA suggested that miR-4670-3p modulates GC proliferation through direct targeting of RORA to modulate the TGF-β/SMAD signaling pathway. Collectively, our findings deepen the understanding of how microRNAs influence sheep fertility and elucidate the molecular mechanism whereby miR-4670-3p promoted GC proliferation through targeting the RORA/TGF-β/SMAD molecular axis.