Background: MicroRNAs play important roles in testicular development and spermatogenesis. Previous research has indicated that the level of miR-200a was significantly upregulated in patients with different spermatogenic impairments. However, the mechanism by which miR-200a regulated spermatogenic impairments remains unclear.
Methods: Leydig cells were treated with triptolide (TP) to mimic spermatogenic impairments. CCK-8 and flow cytometry were used to detect the proliferation and apoptosis in Leydig cells, respectively. In addition, Western blot assay was used to examine ATG7, ATG5, p62 protein levels in MLTC-1 cells.
Results: TP dose-dependently upregulated the expression of miR-200a in MLTC-1 cells. In addition, TP inhibited the proliferation of MLTC-1 cells via inducing apoptosis and oxidative stress; however, these phenomena were notably reversed by miR-200a antagomir. Furthermore, luciferase reporter assay identified that ATG7 was the direct binding target of miR-200a. TP treatment markedly inhibited the activation of autophagy in MLTC-1 cells via inhibition of ATG7. Conversely, downregulation of miR-200a significantly induced autophagy in TP-treated MLTC-1 cells by activation of ATG7. Meanwhile, the cell protective effects of miR-200a against TP were reversed by autophagy inhibitor 3MA, indicating that autophagy plays an important role.
Conclusion: These results indicated that downregulation of miR-200a could protect MLTC-1 cells against TP by inducing autophagy. Therefore, miR-200a might serve as a new therapeutic target for the treatment of male hypogonadism.



中文翻译：

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miR-200a 的下调通过触发自噬保护小鼠睾丸间质细胞免受雷公藤内酯的影响

背景：微小RNA在睾丸发育和精子发生中发挥重要作用。先前的研究表明，在具有不同生精功能障碍的患者中，miR-200a 的水平显着上调。然而，miR-200a 调节生精障碍的机制仍不清楚。
方法： Leydig 细胞用雷公藤甲素 (TP) 处理以模拟生精障碍。CCK-8和流式细胞仪分别用于检测Leydig细胞的增殖和凋亡。此外，采用Western印迹法检测MLTC-1细胞中ATG7、ATG5、p62蛋白水平。
结果：TP 剂量依赖性地上调 MLTC-1 细胞中 miR-200a 的表达。此外，TP通过诱导细胞凋亡和氧化应激抑制MLTC-1细胞的增殖；然而，这些现象被 miR-200a antagomir 显着逆转。此外，荧光素酶报告基因检测发现 ATG7 是 miR-200a 的直接结合靶标。TP 处理通过抑制 ATG7 显着抑制 MLTC-1 细胞中自噬的激活。相反，miR-200a 的下调通过激活 ATG7 在 TP 处理的 MLTC-1 细胞中显着诱导自噬。同时，miR-200a对TP的细胞保护作用被自噬抑制剂3MA逆转，表明自噬发挥了重要作用。
结论：这些结果表明，miR-200a的下调可以通过诱导自噬来保护MLTC-1细胞免受TP的侵害。因此，miR-200a 可能作为治疗男性性腺功能减退症的新治疗靶点。