RNAi-mediated knockdown of INHBB increases apoptosis and inhibits steroidogenesis in mouse granulosa cells

Inhibins are members of the TGFβ superfamily and act as suppressorsof follicle stimulating hormone (FSH) secretion from pituitary glandsvia a negative feedback mechanism to regulate folliculogenesis. Inthis study, the INHBB gene was knocked down by threeRNAi-Ready pSIREN-RetroQ-ZsGreen vector- mediated recombinant plasmidsto explore the effects of INHBB silencing ongranulosa cell (GC) cell cycle, apoptosis and steroid productionin vitro. Quantitative real-time polymerase chainreaction, Western blot, flow cytometry and ELISA were performed toevaluate the role of INHBB in the mouse GC cellcycle, apoptosis and steroid production in vitro. Theresults showed that the relative mRNA and protein expression ofINHBB in mouse GCs can be significantly reduced byRNAi with pshRNA-B1, pshRNA-B2 and pshRNA-B3 plasmids, with pshRNA-B3having the best knockdown efficiency. Downregulation of the expressionof INHBB significantly arrests cells in the G1 phaseof the cell cycle and increases the apoptosis rate in GCs. This wasfurther confirmed by downregulation of the protein expressions ofCyclin D1, Cyclin E and Bcl2, while the protein expression of Bax wasupregulated. In addition, specific downregulation ofINHBB markedly decreased the concentration ofestradiol and progesterone, which was further validated by thedecrease in the mRNA levels of CYP19A1andCYP11A1. These findings suggest that inhibin βB isimportant in the regulation of apoptosis and cell cycle progression ingranulosa cells. Furthermore, the inhibin βB subunit has a role in theregulation of steroid hormone biosynthesis. Evidence is accumulatingto support the concept that inhibin βB is physiologically essentialfor early folliculogenesis in the mouse.