In antral follicles, the transition of proliferative granulosa cells to estradiol-producing is critical for proper oocyte maturation. MicroRNAs are noncoding RNAs that play important roles in ovarian follicular development; however, this has yet to be fully characterized. MicroRNA-21 is significantly higher in granulosa cells isolated from large antral follicles compared to those from small antral follicles. To investigate the function of miR-21, porcine granulosa cells were transfected with miR-21 mimic or miR-21 targeted siRNA. Cells with the miR-21 mimic had higher aromatase expression and estradiol production but decreased WT1 expression. Conversely, cells with the miR-21 siRNA secreted less estradiol and had higher WT1 expression. We hypothesized that miR-21 promotes estradiol production by inhibiting WT1 protein synthesis. We found a potential miR-21 binding site in the 3'UTR of the WT1 transcript and performed a dual-luciferase reporter assay using the WT and mutated 3'UTR. Compared to the negative control, the miR-21 mimic induced a significant decrease in luciferase activity in the WT 3'UTR. This decrease was reversed when the 3'UTR was mutated, suggesting miR-21 targets this site to inhibit WT1 expression. We next transfected porcine granulosa cells with WT1 targeted siRNA and observed a significant increase in aromatase expression and estradiol secretion. We propose that miR-21 represses WT1 expression in granulosa cells to potentially promote aromatase expression and estradiol production. This study offers the first report of a microRNA regulating WT1 expression in granulosa cells and reveals the role of miR-21 in WT1's regulation of estradiol production.
Keywords: WT1; estradiol production; granulosa cell; miR-21; microRNA.