Abstract
MiRNAs regulate follicle development and atresia, steroid production, granulosa cell (GC) proliferation, and apoptosis. However, the target genes and the functioning of novel miRNAs remain unexplored. We reveal the targeting relationship between novel-m0297-5p and WNT5A and the specific regulatory mechanism of GC proliferation in small-tailed Han sheep using whole transcriptomic sequencing. We performed whole transcriptomic sequencing on small-tailed Han sheep ovarian GCs supplemented with 10 ng/mL of transforming growth factor-β1 (TGF-β1) during the early stages. This led to identifying the differential expression of novel-m0297-5p and Wnt family member 5A (WNT5A) and predicting their targeting relationship. Based on this, we hypothesized that TGF-β1 could mediate novel-m0297-5p targeting WNT5A to participate in the proliferation process of GCs in small-tailed sheep. We confirmed the relationship between TGF-β1 and both novel-m0297-5p and WNT5A. The mimicry of novel-m0297-5p inhibited GC activity and proliferation. However, the inhibition of novel-m0297-5p yielded the opposite effect. We validated the binding site for novel m0297-5p within the 3'UTR of WNT5A using dual-luciferase reporter gene. TGF-β1 alleviated the impact induced by the mimicry of novel-m0297-5p on cell viability. Inhibitor co-transfection for both novel-m0297-5p and si-WNT5A suppressed the granulocyte proliferation induced by novel-m0297-5p inhibition. These findings suggest that TGF-β1 can mediate the inhibitory effect of novel-m0297-5p targeting WNT5A on GC proliferation and activity in small-tailed Han sheep. This study provides an experimental basis for research on the biological function of GCs and their impact on follicle development.