Project description:MicroRNAs (miRNAs) have known roles in the post-transcriptional regulation of various biological processes including ovarian follicle development. We have previously identified from human pre-ovulatory granulosa cells miRNAs that are expressed from the intronic regions of two key genes in normal follicular development: FSH receptor (FSHR) and CYP19A1, the latter encoding the aromatase enzyme. In the present study, we aim to identify the targets regulated by those two miRNAs: hsa-miR-548ba and hsa-miR-7973, respectively. The miRNAs of interest were endogenously expressed in KGN cell-line, gene expression changes were analyzed by Affymetrix microarray and confirmed by RT-qPCR. Potential miRNA-regulated sequences were further filtered from the obtained results by bioinformatic target prediction algorithms and validated for direct miRNA:mRNA binding by luciferase reporter assay. Our results verified Leukemia inhibitory factor receptor (LIFR), Phosphatase and tensin homolog (PTEN), Neogenin 1 (NEO1) and SP110 nuclear body protein (SP110) as target genes for hsa-miR-548ba. Hsa-miR-7973 target genes ADAM metallopeptidase domain 19 (ADAM19), Peroxidasin (PXDN) and Formin like 3 (FMNL3) also passed all verification steps. In conclusion we propose that hsa-miR-548ba may be involved in the regulation of follicle growth and activation via LIFR and PTEN. Hsa-miR-7973 may be implicated in the modulation of extracellular matrix and cell-cell interactions. Taken together, our results suggest that those two miRNAs of interest have important regulatory roles in granulosa cells and in follicle development in general.
Project description:To investigate the pathogenesis of diminished ovarian reserve, differentially expressed miRNAs in granulosa cells were constructed through miRCURYTM LNA expression Array.
Project description:The study aimed to uncover the release of miRNAs via EVs and the differential release of these miRNAs from bovine granulosa cells in response to heat stress
Project description:Genome-wide association studies (GWAS) have identified polycystic ovary syndrome (PCOS)-associated loci, including DENND1A encoding a clathrin-binding protein involved in vesicle transport as a guanine nucleotide exchange factor. The effect of DENND1A on reproduction and whether it participates in follicle development disorder and androgen synthesis in PCOS remain unknown. Here, we demonstrated that DENND1A expression increased in ovarian granulosa cells (GCs) of PCOS patients and was positively correlated with serum testosterone concentration. We constructed a systemic transgenic Dennd1a mouse (TG mouse) model, which showed subfertility, irregular estrous cycles, and increased production of testosterone after PMSG stimulation. Moreover, TG mice showed hyporesponsiveness to PMSG with smaller ovary size and less well-developed follicles. Intracellular follicle-stimulating hormone receptor (FSHR) transport was disturbed by overexpression of Dennd1a, which promoted FSHR internalization and inhibited its recycling. Our findings revealed the reproductive function of DENND1A and the underlying mechanisms, which provided new insights into the PCOS pathogenesis and contributing to drug design for PCOS treatment.
Project description:To identify the genes which have relation to aromatase-inhibitor-resistance, we compared the gene expression of aromatase-inhibitor-resistant cell, A3, with that of parent cell line, T-47D.