Hu Sheep Academy, Nanjing Agricultural University, Nanjing, China.
Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, China.
FASEB J. 2022 Sep;36(9):e22467. doi: 10.1096/fj.202200632R.
Although long non-coding RNAs (lncRNAs) are reported to regulate follicular development and reproductive disease pathogenesis, the underlying mechanisms have not been elucidated. In this study, lncRNA expression profiling of different-sized healthy follicles from Hu sheep with different prolificacy revealed 50 613 lncRNAs. Numerous lncRNAs were differentially expressed among different comparison groups. This study characterized one novel transcript, lncRNA-412.25 (from healthy follicles with a diameter of >5 mm), which was predominantly expressed in the high prolificacy group and localized to the cytoplasm of granulosa cells (GCs). LncRNA-412.25 knockdown promoted and inhibited Hu sheep GC apoptosis and proliferation, respectively. Interestingly, lncRNA-412.25 could directly bind to miR-346, which can target the gene of leukemia inhibitory factor (LIF). Knockdown of lncRNA-412.25 promoted GC apoptosis by downregulating LIF expression, where this effect was attenuated by miR-346. Moreover, the miR-346 inhibitor mitigated the lncRNA-412.25 knockdown-induced downregulation of phosphorylated protein of signal transducer and activator of transcription 3 (STAT3), which was validated using immunofluorescence analysis. Our results demonstrated that lncRNA-412.25 regulates GC proliferation and apoptosis in Hu sheep by binding to miR-346 and then activating the LIF/STAT3 pathway. These findings provide novel insights into the mechanisms underlying prolificacy in sheep.
尽管长链非编码 RNA（lncRNA）被报道可以调节卵泡发育和生殖疾病的发病机制，但其中的具体机制尚未阐明。本研究通过对具有不同繁殖力的湖羊不同大小的健康卵泡进行 lncRNA 表达谱分析，共鉴定到 50613 个 lncRNA。在不同的比较组中，许多 lncRNA 存在差异表达。本研究还鉴定到一个新的转录本 lncRNA-412.25（来源于直径>5mm 的健康卵泡），其在高繁殖力组中表达丰度较高，并定位于颗粒细胞（GCs）的细胞质中。lncRNA-412.25 敲低分别促进和抑制了湖羊 GC 的凋亡和增殖。有趣的是，lncRNA-412.25 可以直接与 miR-346 结合，miR-346 可以靶向白血病抑制因子（LIF）的基因。lncRNA-412.25 敲低通过下调 LIF 表达促进 GC 凋亡，而 miR-346 的抑制作用减弱了这一效应。此外，miR-346 抑制剂减轻了 lncRNA-412.25 敲低引起的 STAT3 磷酸化蛋白的下调，这一点通过免疫荧光分析得到了验证。本研究结果表明，lncRNA-412.25 通过与 miR-346 结合，然后激活 LIF/STAT3 通路，调节湖羊 GC 的增殖和凋亡。这些发现为绵羊繁殖力的机制提供了新的见解。
