Li Bo, Hu Xiaopeng, Yang Yanzhou, Zhu Mingyan, Zhang Jiong, Wang Yanrong, Pei Xiuying, Zhou Huchen, Wu Ji
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.
Mol Ther Nucleic Acids. 2019 Sep 6;17:436-447. doi: 10.1016/j.omtn.2019.06.012. Epub 2019 Jun 28.
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.
最近的几项研究揭示了雌性生殖系干细胞（FGSC）分化、增殖和凋亡的调控机制，但自噬等其他生物学过程及其在FGSCs中的机制在很大程度上尚不清楚。使用小分子化合物可能是进一步研究FGSC发育过程中自噬过程和机制的良好方法。在本研究中，我们使用苯并硼唑衍生物ZCL-082处理FGSCs。通过细胞计数试剂盒-8（CCK8）和5-乙炔基-2'-脱氧尿苷（EdU）检测，我们发现ZCL-082可显著降低体外培养的FGSCs的活力、增殖能力和数量。此外，蛋白质印迹分析显示，用ZCL-082处理3小时和6小时后，FGSCs中轻链3β2（LC3B-II）的表达显著增加。同时，聚集体蛋白1（SQSTM1）的表达显著降低。这些结果表明ZCL-082可在体外诱导FGSCs的自噬。关于分子机制，ZCL-082可显著降低生长停滞特异性5（GAS5）长链非编码RNA的表达，该RNA可直接与微小RNA-21a（miR-21a）结合，并在FGSCs中相互负调控。敲低GAS5可诱导FGSCs的自噬，而GAS5过表达则在体外抑制FGSCs的自噬，并挽救由ZCL-082诱导的FGSCs自噬。此外，与对照细胞相比，miR-21a过表达显著增强了FGSCs中LC3B-II蛋白的表达，同时显著降低了程序性细胞死亡蛋白4（PDCD4）和SQSTM1蛋白的表达。抑制miR-21a可显著降低基础或ZCL-082诱导的LC3B-II上调表达，并显著增强PDCD4的表达，同时下调基础或ZCL-082诱导的FGSCs中SQSTM1的表达。此外，GAS5过表达增强了PDCD4的蛋白表达，但敲低GAS5则降低了PDCD4的蛋白表达。综上所述，这些结果表明ZCL-082通过GAS5作为miR-21a的竞争性内源RNA（ceRNA）海绵在FGSCs中诱导自噬。这也表明GAS5/miR-21a轴可能是临床上卵巢早衰的潜在治疗靶点。
