Shi Guanglin, Wang Qian, Zhou Xiaoyu, Li Jun, Liu Hua, Gu Jun, Wang Haiying, Wu Yi, Ding Liang, Ni Songshi, Tang Zhiyuan
Department of Respiratory Medicine, Affiliated Hospital of Nantong University, Nantong 226001, China.
Acta Biochim Biophys Sin (Shanghai). 2017 Apr 1;49(4):302-310. doi: 10.1093/abbs/gmx006.
A number of significant studies in the field of cell biology have revealed another pattern of intracellular signal transduction in which cells transmit information through the dynamics of key signaling molecules. Dynamical properties of p53 have been demonstrated to be the key factor in dictating cell fate, including cell cycle arrest, permanent cell cycle arrest, and cell death. Previous studies showed a negative feedback regulation pathway between SGK1 and p53, but the dynamics of SGK1 have never been reported before. Therefore, we used different dosing strategies of Wogonin to affect SGK1 dynamics and investigate its impact on cell response. Key factors, such as APAF1, BAX, GADD45A, p21, PML, and YPEL3, which are related to cell cycle arrest, senescence, and apoptosis, were measured at different time points after incubation with Wogonin. Western blot and quantitative reverse transcriptase-polymerase chain reaction analysis were used to examine protein and mRNA expression of these genes. In addition, we also used β-galactosidase staining and flow cytometric analysis to further verify the results. It was found that Wogonin inhibited cell viability and downregulated SGK1 protein levels; 20 μM Wogonin could induce non-small-cell lung cancer A549 cells into cell cycle arrest/senescence/apoptosis after 0.5/2/4 h, respectively; and SGK1 dynamics showed significant differences under different cell responses. Together, our findings showed that SGK1 protein dynamics can be an important part of intracellular signaling, directly influencing cellular response decisions.
细胞生物学领域的一些重要研究揭示了另一种细胞内信号转导模式，即细胞通过关键信号分子的动态变化来传递信息。p53的动态特性已被证明是决定细胞命运的关键因素，包括细胞周期停滞、永久性细胞周期停滞和细胞死亡。先前的研究表明SGK1和p53之间存在负反馈调节途径，但SGK1的动态变化此前从未被报道过。因此，我们使用不同的汉黄芩素给药策略来影响SGK1的动态变化，并研究其对细胞反应的影响。在与汉黄芩素孵育后的不同时间点，检测了与细胞周期停滞、衰老和凋亡相关的关键因子，如APAF1、BAX、GADD45A、p21、PML和YPEL3。采用蛋白质印迹法和定量逆转录-聚合酶链反应分析来检测这些基因的蛋白质和mRNA表达。此外，我们还使用β-半乳糖苷酶染色和流式细胞术分析来进一步验证结果。结果发现，汉黄芩素抑制细胞活力并下调SGK1蛋白水平；20 μM汉黄芩素分别在0.5/2/4小时后可诱导非小细胞肺癌A549细胞进入细胞周期停滞/衰老/凋亡状态；并且SGK1的动态变化在不同的细胞反应下表现出显著差异。总之，我们的研究结果表明，SGK1蛋白动态变化可能是细胞内信号传导的重要组成部分，直接影响细胞反应决策。
