miR-378d is Involved in the Regulation of Apoptosis and Autophagy of and E2 Secretion from Cultured Ovarian Granular Cells Treated by Sodium Fluoride

Abstract

Taking excessive sodium fluoride may cause female reproductive dysfunction, but underlying molecular mechanism is unclear. The ovarian granulosa cells are the key endocrine cells releasing reproductive hormones. The miRNAs in the granulosa cells play an important function in regulating reproduction. The aim of this study is to explore the role of miRNAs in granulosa cell apoptosis and autophagy, as well as estradiol (E2) release in response to excessive sodium fluoride. The ovarian granulosa cells (KGN cells) were treated in vitro by different concentrations of sodium fluoride (NaF) for 24 h. The level of estradiol (E2) in the incubation medium was measured by ELISA kits. The total RNA and protein were collected and purified from KGN cells. The expression of miRNAs was detected by the real-time PCR. The signal molecules involved in cell apoptosis and autophagy were detected by the real-time PCR and Western blotting. Six miRNAs in granulosa cells were significantly up- or downregulated by NaF and selected for real-time PCR analysis. The miR-378d was the most significantly upregulated one dose dependently by NaF. It was positively correlated to the extent of apoptosis but negatively correlated to the level of autophagy in KGN cells in response to NaF. In addition, miR-378d promoted E2 release in response to 1 and 2 mM NaF but reduced E2 release in response to 4 and 8 mM NaF treatments. It is concluded that expression of miR-378d in ovarian granulosa cells is negatively correlated to the autophagy and E2 release and positively correlated to cell apoptosis under the influence of NaF.