Ren Siyu, Liu Yuan, Guo Yajing, Zhao Zhihui, Cui Jingjing, Li Mingna, Wang Jiqing
Key Laboratory of Herbivorous Animal Biotechnology, Faculty of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China.
Int J Mol Sci. 2025 Feb 24;26(5):1961. doi: 10.3390/ijms26051961.
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 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 () and predicting their targeting relationship. Based on this, we hypothesized that TGF-β1 could mediate novel-m0297-5p targeting 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 . 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 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 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.
微小RNA（miRNAs）调控卵泡发育与闭锁、类固醇生成、颗粒细胞（GC）增殖及凋亡。然而，新型miRNAs的靶基因及其功能仍未得到探索。我们利用全转录组测序揭示了小尾寒羊中新型-m0297-5p与[Wnt家族成员5A（WNT5A）]之间的靶向关系以及GC增殖的具体调控机制。我们在早期阶段对添加10 ng/mL转化生长因子-β1（TGF-β1）的小尾寒羊卵巢GC进行了全转录组测序。这使得我们鉴定出新型-m0297-5p和Wnt家族成员5A（WNT5A）的差异表达，并预测了它们的靶向关系。基于此，我们推测TGF-β1可介导新型-m0297-5p靶向WNT5A参与小尾寒羊GC的增殖过程。我们证实了TGF-β1与新型-m0297-5p和WNT5A之间的关系。新型-m0297-5p模拟物抑制了GC活性和增殖。然而，抑制新型-m0297-5p则产生相反的效果。我们使用双荧光素酶报告基因验证了WNT5A的3'非翻译区内新型m0297-5p的结合位点。TGF-β1减轻了新型-m0297-5p模拟物对细胞活力的影响。新型-m0297-5p抑制剂与si-WNT5A共转染抑制了新型-m0297-5p抑制诱导的粒细胞增殖。这些发现表明，TGF-β1可介导新型-m0297-5p靶向WNT5A对小尾寒羊GC增殖和活性的抑制作用。本研究为GC生物学功能及其对卵泡发育影响的研究提供了实验依据。
