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MicroRNA-574 Impacts Granulosa Cell Estradiol Production via Targeting TIMP3 and ERK1/2 Signaling Pathway.

ABSTRACT: Estradiol represents a key steroid ovarian hormone that not only plays a vital role in ovarian follicular development but also is associated with many other reproductive functions. Our primary study revealed that miR-574 expression decreased in porcine granulosa cells during development from small to large follicles, and the increase of ERK1/2 phosphorylation accompanies this change. Since it has been well established that the ERK1/2 activity is tightly associated with granulosa cell functions, including ovarian hormone production, we thus further investigate if the miRNA is involved in the regulation of estradiol production in granulosa cells. We found that overexpression of miR-574 decreased phosphorylated ERK1/2 without affecting the level of ERK1/2 protein, and on the other hand, the inhibition of miR-574 increased phosphorylated ERK1/2 level (P<0.05); meanwhile, overexpression of miR-574 increased estradiol production but knockdown of miR-574 decreased estradiol level in granulosa cells. To further identify the potential mechanism involved in the miR-574 regulatory effect, in silico screening was performed and revealed a potential binding site on the 3'UTR region of the tissue inhibitor of metalloproteinase 3 (TIMP3). Our gain-, loss- of function experiments, and luciferase reporter assay confirmed that TIMP3 is indeed the target of miR-574 in granulosa cell. Furthermore, the siRNA TIMP3 knockdown resulted in decreased phosphorylated ERK1/2, and an increase in estradiol production. In contrast, the addition of recombinant TIMP3 increased phosphorylated ERK1/2 level and decreased estradiol production. In summary, our results suggest that the miR-574-TIMP3-pERK1/2 cascade may be one of the pathways by which microRNAs regulate granulosa cell estradiol production.

SUBMITTER: Pan B

PROVIDER: S-EPMC9261285 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:microRNAs are a novel set of small, non-protein-coding nucleotide RNAs that negatively regulate the expression of target mRNAs. miRNA-21 is a microRNA that is highly enriched in endothelial cells. miRNA-21 has been shown to be a potential pro-angiogenic factor in some biological systems. Our previous study showed that the expression of miRNA-21 was up-regulated after spinal cord injury. However, the effect of miRNA-21 on angiogenesis in the spinal cord was unclear. In this study, to understand the role of miRNA-21 on injured endothelial cells exclusively, an oxygen and glucose deprivation model of endothelial cells was constructed, and the up-regulation of miRNA-21 was discovered in this model. An increased level of miRNA-21 by mimics promoted the survival, migration and tube formation of endothelial cells, which simultaneously inhibited tissue inhibitor of metalloproteinase-3 (TIMP3) expression and promoted matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9) expression and secretion. A decreased level of miRNA-21 by antagomir exerted an opposite effect. As is well known, survival, migration and tube formation of endothelial cells are necessary prerequisites for angiogenesis after injury. TIMP3 was validated as a direct target of miRNA-21 by dual-luciferase reporter assay. Silencing with small interfering RNA against TIMP3 promoted tube formation and increased MMP2 and MMP9 expression at the protein level. In vivo, we found that decreased levels of miRNA-21 inhibited angiogenesis after spinal cord injury in rats using synchrotron radiation micro-computed tomography. In summary, these findings suggest that miRNA-21 has a protective effect on angiogenesis by reducing cell death and promoting cell survival, migration and tube formation via partially targeting the TIMP3 by potentially regulating MMP2 and MMP9. TIMP3 is a functional target gene. Identifying the role of miRNA-21 in the protection of angiogenesis might offer a novel therapeutic target for secondary spinal cord injury, in which angiogenesis is indispensable.

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MicroRNA-574 Impacts Granulosa Cell Estradiol Production via Targeting TIMP3 and ERK1/2 Signaling Pathway.

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