C89 Induces Autophagy of Female Germline Stem Cells via Inhibition of the PI3K-Akt Pathway In Vitro.

Xinyue Li,Xiaopeng Hu,Huchen Zhou,Mingyan Zhu,Geng G Tian,Zezhong Li,Ping Cheng,Ji Wu

doi:10.3390/cells8060606

Xinyue Li, Xiaopeng Hu + Show 6 more

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https://doi.org/10.3390/cells8060606

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Journal: Cells	Publication Date: Jun 18, 2019
Citations: 12	License type: CC BY 4.0

Affiliation: Shanghai Jiao Tong University, Ningxia Medical University

Abstract

Postnatal female germline stem cells (FGSCs) are a type of germline stem cell with self-renewal ability and the capacity of differentiation toward oocyte. The proliferation, differentiation, and apoptosis of FGSCs have been researched in recent years, but autophagy in FGSCs has not been explored. This study investigated the effects of the small-molecule compound 89 (C89) on FGSCs and the underlying molecular mechanism in vitro. Cytometry, Cell Counting Kit-8 (CCK8), and 5-ethynyl-2’-deoxyuridine (EdU) assay showed that the number, viability, and proliferation of FGSCs were significantly reduced in C89-treated groups (0.5, 1, and 2 µM) compared with controls. C89 had no impact on FGSC apoptosis or differentiation. However, C89 treatment induced the expression of light chain 3 beta II (LC3BII) and reduced the expression of sequestosome-1 (SQSTM1) in FGSCs, indicating that C89 induced FGSC autophagy. To investigate the mechanism of C89-induced FGSC autophagy, RNA-seq technology was used to compare the transcriptome differences between C89-treated FGSCs and controls. Bioinformatics analysis of the sequencing data indicated a potential involvement of the phosphatidylinositol 3 kinase and kinase Akt (PI3K-Akt) pathway in the effects of C89′s induction of autophagy in FGSCs. Western blot confirmed that levels of p-PI3K and p-Akt were significantly reduced in the C89- or LY294002 (PI3K inhibitor)-treated groups compared with controls. Moreover, we found cooperative functions of C89 and LY294002 in inducing FGSC autophagy through suppressing the PI3K-Akt pathway. Taken together, this research demonstrates that C89 can reduce the number, viability, and proliferation of FGSCs by inducing autophagy. Furthermore, C89 induced FGSC autophagy by inhibiting the activity of PI3K and Akt. The PI3K-Akt pathway may be a target to regulate FGSC proliferation and death.

Highlights

Female germline stem cell (FGSC) lines were successfully established from postnatal mammal and human ovarian tissue in vitro, and the establishment of these lines has opened up new avenues for research [1,2,3,4]
FGSCs were treated with various concentrations of compound 89 (C89) (0.125, 0.25, 0.5, 1, and 2 μM) for 24 and 48 h, and the numbers of viable FGSCs after treatment were counted by a hemocytometer
The number of viable cells was significantly reduced in FGSCs treated with 0.5, 1, and 2 μM C89 compared with controls (Figure 1A–C)

Summary

Introduction

Female germline stem cell (FGSC) lines were successfully established from postnatal mammal and human ovarian tissue in vitro, and the establishment of these lines has opened up new avenues for research [1,2,3,4]. Autophagy is the cellular process of degrading damaged or superfluous organelles and proteins [11,12]. Autophagy occurs constitutively at a basal level and mainly provides housekeeping functions to maintain cellular function. Under stress conditions (e.g., during starvation), autophagy is induced to regenerate nutrients or induce cell death [13,14]. Autophagy can affect the growing status of cells, regulate cell proliferation, or induce cell death.

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