Yu Miaoying, Wei Yinghui, Xu Kui, Liu Shasha, Ma Lei, Pei Yangli, Hu Yanqing, Liu Zhiguo, Zhang Xue, Wang Bingyuan, Mu Yulian, Li Kui
State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.
College of Life Science, Shangrao Normal University, Shangrao, Jiangxi, China.
PeerJ. 2019 Jan 29;7:e6314. doi: 10.7717/peerj.6314. eCollection 2019.
Self-renewal and pluripotency are considered as unwavering features of embryonic stem cells (ESCs). How ESCs regulate the self-renewal and differentiation is a central question in development and regenerative medicine research. Epidermal growth factor receptor (EGFR) was identified as a critical regulator in embryonic development, but its role in the maintenance of ESCs is poorly understood.
Here, EGFR was disrupted by its specific inhibitor AG1478 in mouse ESCs (mESCs), and its self-renewal and pluripotency were characterized according to their proliferation, expression of pluripotency markers, embryoid body (EB) formation, and mRNA expression patterns. We also used another EGFR inhibitor (gefitinib) and RNA interference assay to rule out the possibility of non-specific effects of AG1478.
EGFR inhibition by AG1478 treatment in mESCs markedly reduced cell proliferation, caused cell cycle arrest at G/G phase, and altered protein expressions of the cell cycle regulatory genes (CDK2 (decreased 11.3%) and proliferating cell nuclear antigen (decreased 25.2%)). The immunoreactivities and protein expression of pluripotency factors (OCT4 (decreased 26.9%)) also dramatically decreased, while the differentiation related genes (GATA4 (increased 1.6-fold)) were up-regulated in mESCs after EGFR inhibition. Meanwhile, EGFR inhibition in mESCs disrupted EB formation, indicating its impaired pluripotency. Additionally, the effects observed by EGFR inhibition with another inhibitor gefitinib and siRNA were consistent with those observed by AG1478 treatment in mESCs. These effects were manifested in the decreased expression of proliferative and pluripotency-related genes and the increased expression of genes involved in differentiation. Moreover, RNA-seq analysis displayed that transcript profiling was changed significantly after EGFR inhibition by AG1478. A large number of differentially expressed genes were involved in cell cycle, apoptotic process, epigenetic modification, and metabolic process, which were related to self-renewal and pluripotency, confirming that EGFR deficiency impaired self-renewal and pluripotency in mESCs.
Taken together, our results demonstrated the importance of EGFR in guarding the stemness of mESCs.
自我更新和多能性被认为是胚胎干细胞（ESC）的稳定特性。ESC如何调节自我更新和分化是发育和再生医学研究中的核心问题。表皮生长因子受体（EGFR）被确定为胚胎发育中的关键调节因子，但其在ESC维持中的作用却知之甚少。
在此，EGFR在小鼠胚胎干细胞（mESC）中被其特异性抑制剂AG1478破坏，并根据其增殖、多能性标志物的表达、胚状体（EB）形成和mRNA表达模式来表征其自我更新和多能性。我们还使用了另一种EGFR抑制剂（吉非替尼）和RNA干扰试验来排除AG1478非特异性作用的可能性。
在mESC中用AG1478处理抑制EGFR可显著降低细胞增殖，导致细胞周期停滞在G/G期，并改变细胞周期调节基因（细胞周期蛋白依赖性激酶2（CDK2，降低11.3%）和增殖细胞核抗原（降低25.2%））的蛋白表达。多能性因子（八聚体结合转录因子4（OCT4，降低26.9%））的免疫反应性和蛋白表达也显著降低，而在mESC中抑制EGFR后，与分化相关的基因（GATA结合蛋白4（GATA4，增加1.6倍））上调。同时，在mESC中抑制EGFR会破坏EB形成，表明其多能性受损。此外，用另一种抑制剂吉非替尼和小干扰RNA抑制EGFR所观察到的效果与在mESC中用AG1478处理所观察到的效果一致。这些效果表现为增殖和多能性相关基因的表达降低以及参与分化的基因表达增加。此外，RNA测序分析显示，用AG1478抑制EGFR后转录谱发生了显著变化。大量差异表达基因参与细胞周期、凋亡过程、表观遗传修饰和代谢过程，这些过程与自我更新和多能性相关，证实EGFR缺陷会损害mESC的自我更新和多能性。
综上所述，我们的结果证明了EGFR在维持mESC干性方面的重要性。
