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

Abstract RORA can affect lambing numbers and cell proliferation in sheep. 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, we generated specific overexpression and interference (inhibition) plasmids targeting each molecule. Enhanced expression of miR-4670-3p markedly increased the rate of granulosa cell proliferation, whereas inhibition of miR-4670-3p repressed granulosa cell proliferation. RORA overexpression or inhibition exhibited opposite effects to those mediated by miR-4670-3p on granulosa cell proliferation. Moreover, RORA overexpression also suppressed the expression of TGF-β/SMAD signalling pathway marker genes, whereas RORA inhibition produced the opposite effect. Co-transfection experiments with miR-4670-3p and RORA suggested that miR-4670-3p modulates granulosa cell 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 granulosa cell proliferation through targeting the RORA/TGF-β/SMAD molecular axis.