142 microRNA-17-92 CLUSTER REGULATES BOVINE GRANULOSA CELL FUNCTION BY TARGETING BMPR2 AND PTEN GENES

Abstract

Normal follicular development, especially from the preantral stage until ovulation, is the critical to ensure the release of a developmentally competent oocyte. We have previously shown that among several clusters of microRNAs, microRNA-17-92 cluster (miR-17-5p, miR-19a, miR-20a, and miR-92a) is differentially expressed between bovine granulosa cells (bGC) derived from preovulatory dominant and subordinate follicles. Here, we aimed to investigate the regulatory role of microRNA-17-92 cluster in bGC function. Among the target genes predicted by the miRWalk database, BMPR2 and PTEN genes were experimentally validated using the pmirGLO Dual Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA). The bGC were aspirated from ovaries obtained from a local slaughterhouse. After determining cell viability and concentration using the trypan blue exclusion method, a total 2.5 × 10⁵ bGC per well were seeded into CytoOne 24-well plate in DMEM/F12-Ham medium (Sigma Aldrich Chemie GmbH, Munich, Germany) supplemented with 10% FBS (Gibco BRL USA, Grand Isalnd, NY, USA) and 1% penicillin/streptomycin (Gibco BRL USA). Then, the bGC were cultured at 37°C with 5% CO₂ and O₂. To investigate the role of microRNA-17-92 cluster in bGC function, 100 nM of individual and cluster of microRNA-17-92 mimic, inhibitor, and negative controls were transfected into subconfluent-cultured bGC. The bGC were harvested 48 h post-transfection and used for RNA isolation and subsequent cDNA synthesis and expression analysis of candidate genes using real-time qPCR. Data analysis was performed using the comparative cycle threshold (Ct) method. A cell proliferation assay was performed using CCK-8 kit (Dojindo EU GmbH, Munich, Germany). Based on the cell diameter measurement done using ImageJ 1.48v software (National Institutes for Health, Bethesda, MD, USA), those bGC with diameter >14 µm were categorized as differentiated cells, whereas those with diameter = 14 µm were considered as undifferentiated cells. MicroRNA-17-92 cluster overexpression on bGC reduced both mRNA and protein expression of BMPR2 and PTEN genes, whereas inhibition of microRNA-17-92 cluster increased their expression. Bovine GC transfected with microRNA-17-92 cluster mimic showed higher proliferation activity and decreased rate of differentiation. The opposite phenotype was observed in bGC transfected with microRNA-17-92 cluster inhibitor. Similarly, miRNA-17-92 cluster mimic transfection increased the expression of markers of proliferation, CCND2 and PCNA, and resulted in down-regulation of CYP11A1 and STAR genes as markers of differentiation. The opposite expression pattern was observed after transfection of miRNA-17-92 cluster inhibitors. In conclusion, the miRNA-17-92 cluster members coordinately regulate bGC proliferation and differentiation by targeting the expression of BMPR2 and PTEN genes.