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Sevoflurane inhibits embryonic stem cell self-renewal and subsequent neural differentiation by modulating the let-7a-Lin28 signaling pathway

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Abstract

The commonly used inhalational anesthetic, sevoflurane, can cause toxicity to the central nervous system of the developing fetus. Lin28 has been reported to regulate let-7a, thereby modulating embryo development, neurodegeneration, and even neuron-related tumorigenesis. We demonstrate that pregnant mice receiving sevoflurane treatment during the early stage of pregnancy give birth to fewer offspring presenting a lower birth weight. We have also treated mouse embryonic stem cells (mESCs) with sevoflurane for 6 h and determined that mESCs self-renewal is repressed, and that differentiation is initiated earlier than in controls. We have induced neural differentiation in the treated mESCs and determined that their neurogenesis is weakened. Furthermore, sevoflurane upregulates the level of let-7a, which might repress mESC self-renewal by directly targeting the Lin28 3′-untranslated region. Lin28 overexpression attenuates the influence of sevoflurane or of let-7a on the self-renewal of mESCs and their subsequent neural differentiation. The let-7a inhibitor also abolishes the influence of sevoflurane. Thus, the let-7a-Lin28 pathway is involved in the sevoflurane-induced inhibition of ESC self-renewal and subsequent neurogenesis. Our study demonstrates the molecular mechanism underlying the side effects of sevoflurane during early development, laying the foundation for studies on the safe and reasonable usage of other inhalational anesthetics.

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    Authors and Affiliations

    1. Department of Anesthesiology, The Eye, Ear, Nose and Throat Hospital, Fudan University, Building No.9, 83 Fenyang Road, Xuhui District, Shanghai, 200031, China

      Xiuwen Yi, Yirong Cai, Nan Zhang & Wenxian Li

    2. Department of Anesthesiology, East Hospital, Tongji University School of Medicine, 150 Jimo Road, Shanghai, 200120, China

      Qingxiu Wang

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    1. Xiuwen Yi
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    2. Yirong Cai
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    3. Nan Zhang
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    4. Qingxiu Wang
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    5. Wenxian Li
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    Correspondence to Qingxiu Wang or Wenxian Li.

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    Contributions

    Xiuwen Yi conducted the study, collected the data, and prepared the manuscript. Yirong Cai collected the data, carried out detailed analysis, and acted as archival author. Nan Zhang conducted the study. Qingxiu Wang designed the study. Wenxian Li designed the study and prepared the manuscript.

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    Xiuwen Yi and Yirong Cai contributed equally to this work.

    This research was supported by the Science and Technology Commission of Shanghai Municipality (11ZR1406200) and by the Key Specialty Construction Project of Pudong Health and Family Planning Commission of Shanghai (grant no. PWZz2013-17).

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    Fig. 1

    (related to Fig. 2) Sevoflurane induces apoptosis and decreases cell proliferation and viability of mESCs during neurogenesis. a Detection of mesodermal (Mixi, T, Snail) and endodermal (Gata4, Afp, Apoa1) markers during the induction of neurogenesis on day 14 (ctrl control). For all experiments, n = 4, average ± SD, *P < 0.05, **P < 0.01. b Apoptosis assay during the induction of neurogenesis on day 14. For all experiments, n = 5, average ± SD, **P < 0.01. c Cell viability assay during the induction of neurogenesis on day 14. For all experiments, n = 4, average ± SD, *P < 0.05. d Sevoflurane inhibited the proliferation of mESCs. For all experiments, n = 4, average ± SD. e Apoptosis assay of mESCs cells treated with sevoflurane. For all experiments, n = 5, average ± SD, *P < 0.05. f Cell viability assay of mESCs treated with sevoflurane. For all experiments, n = 5, average ± SD, ***P < 0.01 (GIF 47 kb)

    High resolution image (TIF 8820 kb)

    Fig. 2

    (related to Fig. 3) let-7a inhibitor abrogated the effect of sevoflurane on apoptosis and cell viability of mESCs. a Apoptosis assay of mESCs transfected with let-7a or control (ctrl). For all experiments, n = 5, average ± SD, **P < 0.01. b Cell viability assay of mESCs transfected with let-7a or control. For all experiments, n = 4, average ± SD, ***P < 0.001. c Overexpression of let-7a inhibited the proliferation of mESCs. For all experiments, n = 4, average ± SD. d Apoptosis assay of mESCs transfected with let-7a inhibitor or control. For all experiments, n = 4, average ± SD. e Cell viability assay of mESCs transfected with let-7a inhibitor or control. For all experiments, n = 3, average ± SD. f let-7a inhibitor had no significant influence on cell proliferation. For all experiments, n = 4, average ± SD. g let-7a inhibitor abrogated the effect of sevoflurane on apoptosis in mESCs. For all experiments, n = 5, average ± SD, *P < 0.05. h let-7a inhibitor abrogated the effect of sevoflurane on cell viability in mESCs. For all experiments, n = 3, average ± SD, *P < 0.05. i let-7a inhibitor abrogated the effect of sevoflurane on cell proliferation in mESCs. For all experiments, n = 5, average ± SD. (GIF 82 kb)

    High resolution image (TIF 10681 kb)

    Fig. 3

    (related to Fig. 4) Overexpression of Lin28 restored the function of let-7a or sevoflurane in mESCs. a Overexpression of Lin28 restored the effect of let-7a on apoptosis (ctrl mESCs treated with no sevoflurane and transfected with empty overexpression control vector, OV-ctrl empty overexpression control vector). For all experiments, n = 5, average ± SD, *P < 0.05. b Cell viability assay of mESCs. For all experiments, n = 4, average ± SD, **P < 0.01. c Overexpression of Lin28 restored the effect of sevoflurane on apoptosis. For all experiments, n = 6, average ± SD, *P < 0.05. d Cell viability assay of mESCs transfected with let-7a inhibitor or control. For all experiments, n = 6, average ± SD, **P < 0.01. (GIF 34 kb)

    High resolution image (TIF 13005 kb)

    Fig. 4

    (related to Fig. 5) Sevoflurane regulates apoptosis and cell viability through the let-7a-Lin28 pathway. a Cell apoptosis assay of mESCs in rescue experiments. For all experiments, n = 5, average ± SD, *P < 0.05, **P < 0.01. b Cell viability assay of mESCs in rescue experiments. For all experiments, n = 6, average ± SD, *P < 0.05. c Cell proliferation assay of mESCs in rescue experiments. For all experiments, n = 3, average ± SD. d Cell apoptosis assay of cell derived from the treated mESCs during neurogenesis in rescue experiments. For all experiments, n = 8, average ± SD, *P < 0.05, **P < 0.01. e Cell viability assay of cells derived from the treated mESCs during neurogenesis. For all experiments, n = 5, average ± SD, *P < 0.05, **P < 0.01. (GIF 79 kb)

    High resolution image (TIF 15633 kb)

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    Yi, X., Cai, Y., Zhang, N. et al. Sevoflurane inhibits embryonic stem cell self-renewal and subsequent neural differentiation by modulating the let-7a-Lin28 signaling pathway. Cell Tissue Res 365, 319–330 (2016). https://doi.org/10.1007/s00441-016-2394-x

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