EGFR deficiency leads to impaired self-renewal and pluripotency of mouse embryonic stem cells

Miaoying Yu; Yinghui Wei; Kui Xu; Shasha Liu; Lei Ma; Yangli Pei; Yanqing Hu; Zhiguo Liu; Xue Zhang; Bingyuan Wang; Yulian Mu; Kui Li

doi:10.7717/peerj.6314

EGFR deficiency leads to impaired self-renewal and pluripotency of mouse embryonic stem cells

PeerJ. 2019 Jan 29:7:e6314. doi: 10.7717/peerj.6314. eCollection 2019.

Authors

Miaoying Yu^#^{1

2}, Yinghui Wei^#¹, Kui Xu¹, Shasha Liu¹, Lei Ma^{1

3}, Yangli Pei¹, Yanqing Hu¹, Zhiguo Liu¹, Xue Zhang¹, Bingyuan Wang¹, Yulian Mu¹, Kui Li¹

Affiliations

¹ State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.
² College of Life Science, Shangrao Normal University, Shangrao, Jiangxi, China.
³ College of Life Science, Shihezi University, Shihezi, Xinjiang, China.

^# Contributed equally.

Abstract

Background: 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.

Methods: 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.

Results: EGFR inhibition by AG1478 treatment in mESCs markedly reduced cell proliferation, caused cell cycle arrest at G₀/G₁ 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.

Conclusions: Taken together, our results demonstrated the importance of EGFR in guarding the stemness of mESCs.

Keywords: AG1478; EGFR inhibition; Mouse embryonic stem cells; Pluripotency; Self-renewal.

Grants and funding

This work was funded by the National Natural Science Foundation of China (31402050, 31330074, and 31272416), the Major National Scientific Research Projects (2015CB943101), and the Agricultural Science and Technology Innovation program (ASTIP-IAS05) of CAAS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.