RNAi-mediated knockdown of INHBB increases apoptosis and inhibits steroidogenesis in mouse granulosa cells.
RNAi-mediated knockdown of INHBB increases apoptosis and inhibits steroidogenesis in mouse granulosa cells.
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RNAi 介导的 INHBB 敲除增加小鼠颗粒细胞的凋亡并抑制类固醇生成
DOI：
10.1262/jrd.2014-158
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Yang L
中科院分区：
文献类型：
--
作者：
M'baye M;Hua G;Khan HA;Yang L
抑制素是TGFβ超家族的成员，通过负反馈机制抑制垂体分泌卵泡刺激素（FSH），调节卵泡发生。本研究利用3种RNAi Ready pSIREN-RetroQ-ZsGreen载体介导的重组质粒敲除INHBB基因，探讨INHBB基因沉默对颗粒细胞（GC）细胞周期、细胞凋亡和甾体激素分泌的影响。采用实时荧光定量PCR、Western blot、流式细胞术和ELISA等方法研究了INHBB对小鼠胃癌细胞周期、凋亡和激素分泌的影响。结果表明，pshRNA-B1、pshRNA-B2和pshRNA-B3质粒均能显著抑制小鼠GC中INHBB的相对mRNA和蛋白表达，其中pshRNA-B3的抑制效果最好。INHBB表达的下调显著地将细胞阻滞在细胞周期的G1期，并增加GC中的凋亡率。Cyclin D1、Cyclin E和Bcl 2蛋白表达下调，而Bax蛋白表达上调，进一步证实了这一点。此外，INHBB的特异性下调显著降低了雌二醇和孕酮的浓度，这进一步通过CYP 19 A1和CYP 11 A1的mRNA水平降低得到验证。这些结果表明，在颗粒细胞的凋亡和细胞周期进程的调节中，βB是重要的。此外，β-胡萝卜素βB亚基在类固醇激素生物合成的调节中起作用。越来越多的证据支持这样一种观点，即在小鼠中，卵泡素βB在早期卵泡发生中是生理必需的。
Inhibins are members of the TGFβ superfamily and act as suppressors of follicle stimulating hormone (FSH) secretion from pituitary glands via a negative feedback mechanism to regulate folliculogenesis. In this study, the INHBB gene was knocked down by three RNAi-Ready pSIREN-RetroQ-ZsGreen vector- mediated recombinant plasmids to explore the effects of INHBB silencing on granulosa cell (GC) cell cycle, apoptosis and steroid production in vitro. Quantitative real-time polymerase chain reaction, Western blot, flow cytometry and ELISA were performed to evaluate the role of INHBB in the mouse GC cell cycle, apoptosis and steroid production in vitro. The results showed that the relative mRNA and protein expression of INHBB in mouse GCs can be significantly reduced by RNAi with pshRNA-B1, pshRNA-B2 and pshRNA-B3 plasmids, with pshRNA-B3 having the best knockdown efficiency. Downregulation of the expression of INHBB significantly arrests cells in the G1 phase of the cell cycle and increases the apoptosis rate in GCs. This was further confirmed by downregulation of the protein expressions of Cyclin D1, Cyclin E and Bcl2, while the protein expression of Bax was upregulated. In addition, specific downregulation of INHBB markedly decreased the concentration of estradiol and progesterone, which was further validated by the decrease in the mRNA levels of CYP19A1and CYP11A1. These findings suggest that inhibin βB is important in the regulation of apoptosis and cell cycle progression in granulosa cells. Furthermore, the inhibin βB subunit has a role in the regulation of steroid hormone biosynthesis. Evidence is accumulating to support the concept that inhibin βB is physiologically essential for early folliculogenesis in the mouse.