Zhao Fan, Wang Nan, Yi Yanglei, Lin Pengfei, Tang Keqiong, Wang Aihua, Jin Yaping
Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi, China.
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
PLoS One. 2016 Dec 14;11(12):e0168246. doi: 10.1371/journal.pone.0168246. eCollection 2016.
Luman (also known as LZIP or CREB3) is a transcription factor and a member of the cAMP responsive element-binding (CREB) family proteins. Although Luman has been detected in apoptotic granulosa cells and disorganized atretic bodies, the physiological function of Luman in follicular development has not been reported. Our objective is to determine the role of Luman in folliculogenesis by knocking down Luman expression in mouse GCs (granulosa cells) using shRNA. Luman expression was successfully knocked down in mouse GCs at the mRNA and protein level, as confirmed by real-time quantitative PCR, western blot and immunofluorescence staining, respectively. Knockdown of Luman significantly decreased the concentrations of estradiol (E2) and progesterone (P4) in cell culture medium. Furthermore, Luman knockdown promoted cell proliferation but had no effect on cell apoptosis. To elucidate the regulatory mechanism underlying the effects of Luman knockdown on steroid synthesis and cell cycle, we measured the mRNA and protein expression levels of several related genes. The expression of Star, Cyp19a1, and Cyp1b1, which encode steroidogenic enzymes, was down-regulated, while that of Cyp11a1 and Runx2, which also encode steroidogenic enzymes, was up-regulated. The expression of the cell cycle factors Cyclin A1, Cyclin B1, Cyclin D2, and Cyclin E was significantly up-regulated. Among apoptosis-related genes, only Bcl-2 was down-regulated, while Caspase 3, Bax and p53 were not significantly affected, suggesting that Luman knockdown may regulate cell cycle activity and hormone secretion at the transcriptional and translational level in mouse GCs. The expression of two important genes associated with folliculogenesis in mouse GCs, Has2 and Ptgs2, were also significantly altered by Luman knockdown. In conclusion, the findings of this study indicate that Luman regulates mouse GCs modulation of steroid synthesis, cell cycle activity and other regulators of folliculogenesis.
鲁曼（也称为LZIP或CREB3）是一种转录因子，属于环磷酸腺苷反应元件结合（CREB）家族蛋白。尽管在凋亡的颗粒细胞和紊乱的闭锁小体中检测到了鲁曼，但尚未报道其在卵泡发育中的生理功能。我们的目标是通过使用短发夹RNA（shRNA）敲低小鼠颗粒细胞（GCs）中的鲁曼表达，来确定鲁曼在卵泡发生中的作用。通过实时定量PCR、蛋白质免疫印迹和免疫荧光染色分别证实，鲁曼表达在小鼠颗粒细胞的mRNA和蛋白质水平上成功被敲低。敲低鲁曼显著降低了细胞培养基中雌二醇（E2）和孕酮（P4）的浓度。此外，敲低鲁曼促进了细胞增殖，但对细胞凋亡没有影响。为了阐明敲低鲁曼对类固醇合成和细胞周期影响的调控机制，我们检测了几个相关基因的mRNA和蛋白质表达水平。编码类固醇生成酶的Star、Cyp19a1和Cyp1b1的表达下调，而同样编码类固醇生成酶的Cyp11a1和Runx2的表达上调。细胞周期因子细胞周期蛋白A1、细胞周期蛋白B1、细胞周期蛋白D2和细胞周期蛋白E的表达显著上调。在凋亡相关基因中，只有Bcl-2下调，而半胱天冬酶3、Bax和p53没有受到显著影响，这表明敲低鲁曼可能在转录和翻译水平上调节小鼠颗粒细胞的细胞周期活性和激素分泌。小鼠颗粒细胞中与卵泡发生相关的两个重要基因Has2和Ptgs2的表达也因敲低鲁曼而显著改变。总之，本研究结果表明，鲁曼调节小鼠颗粒细胞对类固醇合成、细胞周期活性及卵泡发生其他调节因子的调控。
