Wei Chaofeng, Xiang Shan, Yu Yi, Song Jingyan, Zheng Mingming, Lian Fang
Master of Gynecology in Traditional Chinese Medicine, College of Traditional Chinese Medicine, Shandong University of Traditional Chinese Medicine, Jinan, Shandong, China.
First College of Clinical Medicine, Shandong University of Traditional Chinese Medicine, Jinan, Shandong, China.
Mol Reprod Dev. 2021 Apr;88(4):251-260. doi: 10.1002/mrd.23457. Epub 2021 Mar 10.
In our earlier study, we showed that the expression of microRNA-221-3p (miR-221-3p) was significantly lower in women of advanced age with diminished ovarian reserve (DOR) compared with young women with normal ovarian reserve (NOR). Therefore, in this study, we aimed to explore how miR-221-3p regulates apoptosis of granulosa cells and the pathogenesis of DOR. Bioinformatics prediction and dual-luciferase reporter assay were conducted to identify the target gene of miR-221-3p. miR-221-3p expression was manipulated by transfecting KGN cells with miR-221-3p mimics, inhibitor, and negative control. Following transfection, apoptosis of granulosa cells was determined by flow cytometry, and the expression of the target gene was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis (WB). In addition, the expression of the target gene in granulosa cells of DOR patients and NOR patients was measured. miR-221-3p were found to directly bind the 3' untranslated region of Forkhead box O1 (FOXO1). Transfection with miR-221-3p mimics significantly decreased the apoptosis rate of KGN cells compared with transfection with miR-221-3p inhibitors. The expression level of miR-221-3p was negatively correlated with the messenger RNA and protein levels of the FOXO1 gene. Besides, FOXO1 expression was upregulated in DOR patients. In conclusion, these results provide evidence that downregulation of miR-221-3p expression promotes apoptosis of granulosa cells by upregulating FOXO1 expression, thus serving an important role in DOR pathogenesis.
在我们早期的研究中，我们发现与卵巢储备功能正常（NOR）的年轻女性相比，卵巢储备功能减退（DOR）的高龄女性中微小RNA-221-3p（miR-221-3p）的表达显著降低。因此，在本研究中，我们旨在探讨miR-221-3p如何调节颗粒细胞凋亡以及DOR的发病机制。通过生物信息学预测和双荧光素酶报告基因检测来鉴定miR-221-3p的靶基因。用miR-221-3p模拟物、抑制剂和阴性对照转染KGN细胞来调控miR-221-3p的表达。转染后，通过流式细胞术测定颗粒细胞凋亡情况，并通过定量实时聚合酶链反应（qRT-PCR）和蛋白质免疫印迹分析（WB）检测靶基因的表达。此外，还检测了DOR患者和NOR患者颗粒细胞中靶基因的表达。发现miR-221-3p直接结合叉头框O1（FOXO1）的3'非翻译区。与用miR-221-3p抑制剂转染相比，用miR-221-3p模拟物转染显著降低了KGN细胞的凋亡率。miR-221-3p的表达水平与FOXO1基因的信使核糖核酸和蛋白质水平呈负相关。此外，DOR患者中FOXO1表达上调。总之，这些结果表明，miR-221-3p表达下调通过上调FOXO1表达促进颗粒细胞凋亡，从而在DOR发病机制中起重要作用。
