MicroRNAs expressed from FSHR and aromatase genes target important ovarian functions.. MicroRNAs expressed from FSHR and aromatase genes target important ovarian functions.

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. Overall design: For Affymetrix microarray miRNA mimics hsa-miR-548ba, hsa-miR-7973 and control miRNA cel-miR-39-3p were transfected with previously optimized conditions: 12.5nM concentration and 72 hours. Each miRNA was transfected in number of four parallel samples.