Key Lab of Environment and Health, School of Public Health, Xuzhou Medical University, 209 Tong-Shan Road, Xuzhou, Jiangsu 221004, China; Key Laboratory of Human Genetics and Environmental Medicine, Xuzhou Medical University, Xuzhou, Jiangsu 221004, China.
Key Lab of Environment and Health, School of Public Health, Xuzhou Medical University, 209 Tong-Shan Road, Xuzhou, Jiangsu 221004, China; Key Laboratory of Human Genetics and Environmental Medicine, Xuzhou Medical University, Xuzhou, Jiangsu 221004, China.
Reprod Toxicol. 2023 Aug;119:108414. doi: 10.1016/j.reprotox.2023.108414. Epub 2023 May 26.
Cypermethrin (CYP) has been identified as one kind of endocrine-disrupting chemicals (EDCs) to induce male reproduction damage. This study aimed to investigate the effects and mechanisms of miR-30a-5p on CYP induced apoptosis of TM4 mouse Sertoli cells in vitro. In the present study, 0 μM, 10 μM, 20 μM, 40 μM and 80 μM CYP were used to treat TM4 cells for 24 h. The apoptosis of TM4 cells, the expression level of miR-30a-5p, the protein expressions and the interaction between miR-30a-5p and KLF9 were detected by flow cytometry, quantitative Real-Time PCR, Western blot and luciferase reporter assays. CYP induced apoptosis of TM4 cells, inhibited expression of miR-30a-5p in TM4 cells, and overexpression of miR-30a-5p partially recovered CYP induced cells apoptosis. Furthermore, KLF9 was a potential downstream target of miR-30a-5p predicted by publicly available databases. KLF9 expression level in TM4 cells was significantly elevated after treatment with CYP, and the induction was inhibited by miR-30a-5p mimics transfection. Meanwhile, dual-luciferase reporter assay demonstrated that miR-30a-5p directly targeted KLF9-3'UTR. Moreover, in the presence of CYP, the apoptosis regulator p53 expression was also increased in TM4 cells. Overexpression miR-30a-5p or down-regulation of KLF9 both attenuated the induction of CYP on p53 expression. Overall, the present study demonstrated that miR-30a-5p regulated CYP induced TM4 cells apoptosis by targeting KLF9/p53 axis.
氯菊酯（CYP）已被确定为一种内分泌干扰化学物质（EDCs），可诱导雄性生殖损伤。本研究旨在探讨 miR-30a-5p 对 CYP 诱导 TM4 小鼠支持细胞体外凋亡的影响及其机制。在本研究中，用 0μM、10μM、20μM、40μM 和 80μM CYP 处理 TM4 细胞 24h。通过流式细胞术、定量实时 PCR、Western blot 和荧光素酶报告基因检测，检测 TM4 细胞的凋亡、miR-30a-5p 的表达水平、蛋白表达以及 miR-30a-5p 与 KLF9 的相互作用。CYP 诱导 TM4 细胞凋亡，抑制 TM4 细胞中 miR-30a-5p 的表达，过表达 miR-30a-5p 部分恢复了 CYP 诱导的细胞凋亡。此外，公共数据库预测 KLF9 是 miR-30a-5p 的潜在下游靶基因。CYP 处理后 TM4 细胞中 KLF9 表达水平显著升高，miR-30a-5p 模拟物转染抑制了诱导作用。同时，双荧光素酶报告基因检测表明 miR-30a-5p 可直接靶向 KLF9-3'UTR。此外，在 CYP 存在的情况下，凋亡调节因子 p53 在 TM4 细胞中的表达也增加。过表达 miR-30a-5p 或下调 KLF9 均可减弱 CYP 对 p53 表达的诱导作用。总之，本研究表明，miR-30a-5p 通过靶向 KLF9/p53 轴调节 CYP 诱导的 TM4 细胞凋亡。
