Abstract
Although miR-27b-3p has been evidenced to regulate the proliferation, apoptosis, and differentiation of a variety of mammalian cell types, its actions and mechanisms on ovarian cell steroidogenesis remains largely unknown in both mammalian and avian species. In this study, we aimed to determine the expression profiles of miR-27b-3p in granulosa cell layers during goose ovarian follicle development and to reveal its actions on estrogen (E(2)) secretion of goose granulosa cells as well as the underlying regulatory mechanisms. It was observed that miR-27b-3p was ubiquitously expressed throughout follicle development but exhibited much higher levels in hierarchical- than in prehierarchical follicles. In cultured granulosa cells from the fourth through second largest preovulatory (F4-F2) follicles of goose, up- and downregulation of miR-27b-3p by using its mimic and inhibitor significantly decreased and increased E(2) secretion, respectively. Meanwhile, the mRNA levels of STAR and CYP19A1 were significantly reduced while those of CYP11A1 and 3βHSD were elevated in the mimic-transfected granulosa cells. By comparison, downregulation of miR-27b-3p enhanced the mRNA levels of STAR but had no significant effects on those of CYP19A1, CYP11A1, and 3βHSD. Results from bioinformatic prediction and luciferase reporter assay demonstrated that CYP1B1 was a downstream target of miR-27b-3p. Although the siRNA-mediated downregulation of CYP1B1 did not significantly change E(2) secretion by goose granulosa cells, it reduced the mRNA levels of STAR and CYP19A1 as well as those of LKB1 and AMPKα, which are involved in the AMPK signaling pathway. Taken together, these data suggest that miR-27b-3p plays an inhibitory role in E(2) secretion by goose F4-F2 granulosa cells, at least in part, by targeting CYP1B1 through the AMPK signaling pathway.