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. 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.