College of Animal Science and Technology, Key Laboratory of Animal Genetic, Breeding and Reproduction in Shaanxi Province, Northwest A & F University, Yangling, China.
Faculty of Medical and Chemical Engineering, Xianyang Vocational Technical College, Xi'an, China.
Reproduction. 2019 Nov;158(5):441-452. doi: 10.1530/REP-19-0285.
It is well documented that granulosa cell apoptosis is the main reason for follicular atresia and death; however, increasing evidence suggests that autophagy plays an important role in the fate of granulosa cells. miR-21-3p regulates many fundamental biological processes and is pivotal in the autophagy of tumor cells; nevertheless, the autophagy in cattle ovary and how miR-21-3p regulates the follicular cells is unknown. In this study, we aimed to elucidate the autophagy and the role of miR-21-3p in cattle ovary using bovine primary ovarian granulosa cells (BGCs). The results showed the autophagy for the first time in BGCs in large follicle according to autophagic gene transcript of LC3, BECN-1, ATG3, protein expression of LC3, P62 and LC3 puncta, a standard marker for autophagosomes. miR-21-3p was identified as a novel miRNA that repressed BGCs autophagy according to the results from plasmids transfection of miR-21-3p mimics and inhibitor. Meanwhile, VEGFA was confirmed to be a validated target of miR-21-3p in BGCs using luciferase reporter assays and the results of VEGFA expression decreased with transfection of miR-21-3p mimics, while it increased with transfection of miR-21-3p inhibitor. In addition, small interference-mediated knockdown of VEGFA significantly inhibits BGCs autophagy signaling; however, overexpression of VEGFA in BGCs promoted autophagy in the presence of miR-21-3p. Finally, the results of AKT and its phosphorylation suggested that miR-21-3p suppressed VEGFA expression through downregulating AKT phosphorylation signaling. In summary, this study demonstrates that miR-21-3p inhibits BGCs autophagy by targeting VEGFA and attenuating PI3K/AKT signaling.
已有大量文献表明，颗粒细胞凋亡是卵泡闭锁和死亡的主要原因；然而，越来越多的证据表明自噬在颗粒细胞命运中起着重要作用。miR-21-3p 调节许多基本的生物学过程，在肿瘤细胞的自噬中起着关键作用；然而，牛卵巢中的自噬以及 miR-21-3p 如何调节卵泡细胞尚不清楚。在这项研究中，我们旨在使用牛原代卵巢颗粒细胞（BGCs）阐明牛卵巢中的自噬以及 miR-21-3p 的作用。结果首次显示，根据 LC3、BECN-1、ATG3 的自噬基因转录物、LC3、P62 和 LC3 斑点的蛋白表达，在大卵泡中的 BGCs 中出现了自噬，LC3 斑点是自噬体的标准标记。根据 miR-21-3p 模拟物和抑制剂的质粒转染结果，miR-21-3p 被鉴定为一种新的 miRNA，可抑制 BGCs 的自噬。同时，通过荧光素酶报告基因检测和 VEGFA 表达降低的结果证实，VEGFA 是 BGCs 中 miR-21-3p 的有效靶标，而 miR-21-3p 模拟物的转染则增加了 VEGFA 的表达。此外，小干扰介导的 VEGFA 敲低可显著抑制 BGCs 的自噬信号；然而，在 miR-21-3p 存在的情况下，BGCs 中 VEGFA 的过表达促进了自噬。最后，AKT 及其磷酸化的结果表明，miR-21-3p 通过下调 AKT 磷酸化信号抑制 VEGFA 的表达。综上所述，本研究表明，miR-21-3p 通过靶向 VEGFA 并减弱 PI3K/AKT 信号抑制 BGCs 的自噬。
