Zhuo Lifan, Zhou Yue, Tian Jia, Li Yan, Xie Zhiyuan, Pei Chengbin, Yan Bei, Ma Lianghong
Institute of Medical Sciences, Ningxia Human Sperm Bank, General Hospital of Ningxia Medical University, Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, Yinchuan, 750004, China.
Institute of Medical Sciences, Ningxia Human Sperm Bank, General Hospital of Ningxia Medical University, Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, Yinchuan, 750004, China.
Theriogenology. 2023 Nov;211:56-64. doi: 10.1016/j.theriogenology.2023.07.008. Epub 2023 Jul 10.
MicroRNAs (miRNAs) play a crucial role in regulating various physiological processes, including cell differentiation, proliferation, and apoptosis. However, their specific functions in response to heat stress are not fully understood. This study aimed to investigate the regulatory effects of miR-199a-3p on the proliferation of heat stress-treated spermatogonial stem cells (SSCs). SSCs were isolated from mouse testes and cultured in vitro to identify marker molecules. Lentiviruses carrying miR-199a-3p-over, miR-199a-3p-inhibit, and ID4-over constructs were generated for stable transfection. Luciferase assay was employed to confirm the targeting relationship between miR-199a-3p and ID4. An in vitro SSCs heat stress model was established, and the miR-199a-3p-inhibit and ID4-over groups were included. Cellular proliferation was assessed using CCK-8, EdU, and cell cycle analysis methods after heat stress. Expression levels of miR-199a-3p and ID4 were evaluated by western blotting and qRT-PCR. The results demonstrated that miR-199a-3p-over inhibited SSCs proliferation, while ID4-over promoted an increase in SSCs number. Luciferase assay confirmed the regulatory effect of miR-199a-3p on ID4 expression. Moreover, after heat stress treatment, miR-199a-3p-inhibit and ID4-over enhanced SSCs proliferation compared to the control group. These findings suggest that miR-199a-3p modulates SSCs proliferation by targeting ID4, especially under heat stress conditions.
微小RNA（miRNA）在调节包括细胞分化、增殖和凋亡在内的各种生理过程中发挥着关键作用。然而，它们在应对热应激时的具体功能尚未完全明确。本研究旨在探讨miR-199a-3p对热应激处理后的精原干细胞（SSC）增殖的调节作用。从小鼠睾丸中分离出SSC并在体外培养以鉴定标记分子。构建携带miR-199a-3p过表达、miR-199a-3p抑制和ID4过表达构建体的慢病毒用于稳定转染。采用荧光素酶测定法确认miR-199a-3p与ID4之间的靶向关系。建立体外SSC热应激模型，并纳入miR-199a-3p抑制组和ID4过表达组。热应激后，使用CCK-8、EdU和细胞周期分析方法评估细胞增殖。通过蛋白质免疫印迹和qRT-PCR评估miR-199a-3p和ID4的表达水平。结果表明，miR-199a-3p过表达抑制SSC增殖，而ID4过表达促进SSC数量增加。荧光素酶测定法证实了miR-199a-3p对ID4表达的调节作用。此外，热应激处理后，与对照组相比，miR-199a-3p抑制组和ID4过表达组增强了SSC增殖。这些发现表明，miR-199a-3p通过靶向ID4调节SSC增殖，尤其是在热应激条件下。
