GAS5/miR-21 Axis as a Potential Target to Rescue ZCL-082-Induced Autophagy of Female Germline Stem Cells In Vitro

Bo Li; Xiaopeng Hu; Yanzhou Yang; Mingyan Zhu; Jiong Zhang; Yanrong Wang; Xiuying Pei; Huchen Zhou; Ji Wu

doi:10.1016/j.omtn.2019.06.012

GAS5/miR-21 Axis as a Potential Target to Rescue ZCL-082-Induced Autophagy of Female Germline Stem Cells In Vitro

Mol Ther Nucleic Acids. 2019 Sep 6:17:436-447. doi: 10.1016/j.omtn.2019.06.012. Epub 2019 Jun 28.

Authors

Bo Li¹, Xiaopeng Hu², Yanzhou Yang¹, Mingyan Zhu³, Jiong Zhang³, Yanrong Wang¹, Xiuying Pei¹, Huchen Zhou⁴, Ji Wu⁵

Affiliations

¹ Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, Yinchuan 750004, China.
² Key Laboratory for the Genetics of Developmental & Neuropsychiatric Disorders (Ministry of Education), Bio-X Institutes, Shanghai Jiao Tong University, Shanghai 200240, China.
³ State Key Laboratory of Microbial Metabolism, School of Pharmacy, Shanghai Jiao Tong University, 200240 Shanghai, China.
⁴ State Key Laboratory of Microbial Metabolism, School of Pharmacy, Shanghai Jiao Tong University, 200240 Shanghai, China. Electronic address: hczhou@sjtu.edu.cn.
⁵ Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, Yinchuan 750004, China; Key Laboratory for the Genetics of Developmental & Neuropsychiatric Disorders (Ministry of Education), Bio-X Institutes, Shanghai Jiao Tong University, Shanghai 200240, China. Electronic address: jiwu@sjtu.edu.cn.

Abstract

Several studies have recently revealed the regulatory mechanisms underlying female germline stem cell (FGSC) differentiation, proliferation, and apoptosis, but other biological processes such as autophagy and its mechanism in FGSCs are largely unclear. The use of small chemical compounds may be a good approach to further investigate the process and mechanism of autophagy in FGSC development. In this study, we used ZCL-082, a derivative of benzoxaboroles, to treat FGSCs. Using a cell counting kit-8 (CCK8) and 5-ethynyl-2'-deoxyuridine (EdU) assays, we found that ZCL-082 could significantly reduce the viability, proliferation, and number of FGSCs in vitro. Moreover, western blotting revealed that the expression of light chain 3 beta 2 (LC3B-II) in FGSCs was significantly increased after treatment with ZCL-082 for 3 and 6 h. Meanwhile, the expression of sequestosome-1 (SQSTM1) was significantly decreased. These results suggested that ZCL-082 can induce autophagy of FGSCs in vitro. Regarding the molecular mechanism, ZCL-082 could significantly reduce the expression of growth arrest-specific 5 (GAS5) long non-coding RNA, which could directly bind to microRNA-21a (miR-21a) and negatively regulate each other in FGSCs. Knockdown of GAS5 induced the autophagy of FGSCs, while GAS5 overexpression inhibited the autophagy of FGSCs in vitro and rescued FGSC autophagy induced by ZCL-082. Additionally, overexpression of miR-21a significantly enhanced LC3B-II protein expression while significantly reducing the expression of programmed cell death protein 4 (PDCD4) and SQSTM1 protein in FGSCs compared with control cells. The inhibition of miR-21a significantly reduced the basal or ZCL-082-induced upregulated expression of LC3B-II, and it significantly enhanced the expression of PDCD4 while downregulating the basal or ZCL-082-induced expression of SQSTM1 in FGSCs. Furthermore, the overexpression of GAS5 enhanced the protein expression of PDCD4, but knockdown of GAS5 reduced the protein expression of PDCD4. Taken together, these results suggested that ZCL-082 induced autophagy through GAS5 functioning as a competing endogenous RNA (ceRNA) sponge for miR-21a in FGSCs. It also suggested that the GAS5/miR-21a axis may be a potential therapeutic target for premature ovarian failure in the clinic.