摘要
MicroRNAs (miRNAs)已经发现在卵巢卵泡发育过程中起着关键作用。但是,关于哺乳动物卵巢中miRNA基因自身的表达调控还知之甚少。本实验室之前报道miR-224通过靶向Smad4参与转化生长因子-β1(TGF-β1)介导的卵泡颗粒细胞生长和雌二醇生成。本研究中,miR-224在颗粒细胞中表达的转录调控被进一步研究。我们的结果显示抑癌基因p53和NF-κB家族p65抑制TGF-β1诱导的pri-miR-224在颗粒细胞中的表达。核质分离和染色质免疫共沉淀实验证实TGF-β1能够诱导p53和p65由胞质向胞核转移和促进p53和p65结合在GABAA受体ε亚基(GABRE)(miR-224宿主基因)近端启动子区域上。p53和p65协同转录抑制GABRE启动子活性。进一步实验发现颗粒细胞中p53和p65可以通过抑制下游miR-224来上调SMAD4表达,这至少部分影响了miR-224和SMAD4对于颗粒细胞增殖和雌二醇分泌的效应。
另外,本实验室之前的研究显示miR-224靶向于在排卵过程中对与卵丘扩展非常关键的基因正五聚蛋白3(Ptx3), TGF-β1可以促进小鼠卵巢壁颗粒细胞和卵丘卵母细胞复合物(COCs)中PTX3表达。本研究中,通过构建腺病毒介导的miR-224过表达载体(/Ad-miR-224),在体内和体外进一步检测miR-224对于排卵的影响。体外研究证实,miR-224通过降低表皮生长因子(EGF)诱导COCs的PTX3分泌来抑制卵丘扩展。体内研究也显示,卵巢囊内注射Ad-miR-224可以降低PTX3表达和抑制排卵,这些导致了胚胎着床和子代出生的数量减少。
综上所述,我们的结果为转录因子通过相互作用调控miRNA表达来参与颗粒细胞增殖和功能提供了新的数据。而且,miR-224可以通过靶向Ptx3影响排卵和接下来的胚胎发育,这提示miRNAs对于治疗排卵障碍相关的不孕或者设计新的避孕药具有潜在的价值。
MicroRNAs (miRNAs) have been indicated to play key roles in ovarian follicular development. However, little is known about how the miRNA gene expression itself is regulated in the mammalian ovary. We previously reported that miR-224is involved in Transforming growth factor-β1(TGF-β1)-mediated mouse preantral granulosa cells (mpGCs) growth and estradiol (E2) production by targeting Smad4. Here, the transcriptional regulation of miR-224expression in GCs was further investigated. Our results showed that both the tumor suppressor gene p53and nuclear factor-κB (NF-κB) p65subunit suppressed the TGF-β1-induced increase in pri-miR-224expression in mpGCs. Separation of cytosolic/nuclear fractions and ChIP assays demonstrated that TGF-β1induced the translocation of p53and p65from the cytoplasm to the nucleus and enhanced the binding of p53and p65to the proximal promoter region of GABAA receptor ε subunit (GABRE)(miR-224host gene). p53and p65transcriptionally cooperated to inactivate the GABRE promoter. Furthermore, p53/p65could up-regulate SMAD4expression by inhibiting its target miR-224in mpGCs which contributed, at least partially, to the effects of miR-224and SMAD4on GC proliferation and E2release.
In addition, our previous study showed that miR-224was found to target pentraxin3(Ptx3), a gene critical for cumulus expansion during ovulation, and PTX3was up-regulated in mouse mural GCs (muGCs) and cumulus-oocyte complexes (COCs) by TGF-β1treatment. Here, the effect of miR-224during ovulation was further examined in vitro and in vivo by construction of an adenovirus-mediated expression vector for miR-224(Ad-miR-224). In vitro studies demonstrated that miR-224could perturb cumulus expansion in EGF-stimulated COCs by decreasing PTX3secretion. In vivo studies also showed that injection of Ad-miR-224into ovarian bursa decreased PTX3expression and disrupted ovulation, which led to a decreased number of implantation sites and offspring being born.
In conclusion, our results provide new data about the interplay between transcription factors involved in GC proliferation and function by cooperatively regulating miRNA expression. Furthermore, miR-224may affect ovulation and subsequent embryo development by targeting Ptx3, suggesting potential roles for miRNAs in offering new treatments for ovulation disorder-associated infertility, or, conversely, designing new contraceptives.
引文
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