MicroRNA-132 promotes estradiol synthesis in ovarian granulosa cells via translational repression of Nurr1.
MicroRNA-132 promotes estradiol synthesis in ovarian granulosa cells via translational repression of Nurr1.
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MicroRNA-132 通过 Nurr1 的翻译抑制促进卵巢颗粒细胞中雌二醇的合成
DOI：
10.1186/s12958-015-0095-z
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发表时间：
2015-08-19
期刊：
影响因子：
--
通讯作者：
Hu Y
中科院分区：
文献类型：
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作者：
Wu S;Sun H;Zhang Q;Jiang Y;Fang T;Cui I;Yan G;Hu Y
破坏原合成是哺乳动物卵巢的重要功能。雌激素在卵泡发育、卵母细胞成熟和子宫内膜增殖等许多生物学过程中发挥重要作用，雌激素合成功能障碍是多囊卵巢综合征和卵巢早衰的重要因素。促卵泡激素触发的经典信号级联反应通过上调颗粒细胞（GCs）中的Cyp19a1诱导雌激素合成。本研究旨在确定microRNA-132 （miR-132）对GCs中雌二醇合成的影响。方法采用卵泡穿刺法从21日龄未成熟ICR小鼠卵巢中采集小鼠原代GCs。用稳定的环磷酸腺苷类似物8-Br-cAMP培养和处理GCs，或用miR-132模拟物、nurr1特异性小干扰RNA寡核苷酸和Flag-Nurr1质粒转染GCs。用自动化学发光法测定培养基中雌二醇和黄体酮的浓度。采用实时荧光定量PCR和western blot检测miR-132对GCs中Cyp19a1、Cyp11a1和孤儿核受体nurr1表达的影响。通过荧光素酶报告基因试验进一步验证了miR-132通过其3'-非翻译区直接抑制Nurr1。结果8-Br-cAMP作用48 h后，miR-132在培养小鼠GCs中的表达水平显著升高。小鼠GCs过表达miR-132后雌二醇的合成增加。real-time PCR结果显示miR-132显著诱导Cyp19a1的表达。Nurr1是一种抑制Cyp19a1表达的孤儿核受体，被发现是miR-132的直接靶点。荧光素酶检测和Western blot显示，Nurr1被miR-132抑制。敲低Nurr1主要提高雌二醇的合成，部分减弱miR-132诱导的雌二醇升高，Flag-Nurr1的异位表达消除了miR-132对小鼠GCs中雌二醇合成的刺激作用。结论研究结果表明miR-132参与cAMP信号通路，并通过抑制卵巢GCs中Nurr1的翻译促进雌二醇合成。
BackgroundEstrogen synthesis is an important function of the mammalian ovary. Estrogen plays important roles in many biological processes, including follicular development, oocyte maturation and endometrial proliferation, and dysfunctions in estrogen synthesis contribute to the development of polycystic ovary syndrome and premature ovarian failure. Classical signaling cascades triggered by follicle-stimulating hormone induce estrogen synthesis via the upregulation of Cyp19a1 in granulosa cells (GCs). This study aimed to determine the effect of microRNA-132 (miR-132) on estradiol synthesis in GCs.MethodsPrimary mouse GCs were collected from ovaries of 21-day-old immature ICR mice through follicle puncture. GCs were cultured and treated with the stable cyclic adenosine monophosphate analog 8-Br-cAMP or transfected with miR-132 mimics, Nurr1-specific small interfering RNA oligonucleotides and Flag-Nurr1 plasmids. Concentrations of estradiol and progesterone in culture medium were determined by an automated chemiluminescence-based assay. Quantitative real time PCR and western blot were performed to identify the effect of miR-132 on Cyp19a1, Cyp11a1 and an orphan nuclear receptor-Nurr1 expression in GCs. Direct suppression of Nurr1 via its 3'-untranslated region by miR-132 were further verified using luciferase reporter assays.ResultsThe expression level of miR-132 in cultured mouse GCs was significantly elevated during 48 h of treatment with 8-Br-cAMP. The synthesis of estradiol increased after the overexpression of miR-132 in mouse GCs. The real-time PCR results demonstrated that miR-132 induced the expression of Cyp19a1 significantly. Nurr1, an orphan nuclear receptor that suppresses Cyp19a1 expression, was found to be a direct target of miR-132. Nurr1 was suppressed by miR-132, as indicated by a luciferase assay and Western blotting. The knockdown of Nurr1 primarily elevated the synthesis of estradiol and partially attenuated the miR-132-induced estradiol elevation, and the ectopic expression of Flag-Nurr1 abrogated the stimulatory effect of miR-132 on estradiol synthesis in mouse GCs.ConclusionsOur findings suggest that miR-132 is involved in the cAMP signaling pathway and promotes estradiol synthesis via the translational repression of Nurr1 in ovarian GCs.