Study On The Molecular Mechanism Of MiRNA-224Expression And Regulation In Mouse Follicular Development

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.