Abstract：
Late-onset hypogonadism (LOH) is an age-related syndrome characterized by deficiency of serum testosterone produced by Leydig cells. Previous evidence suggested that microRNA (miR)-361-3p can serve as a promising biomarker for LOH. Nonetheless, its detailed function and molecular mechanism in LOH remain unclarified. The 24-month-old male mice were selected as an animal LOH model, and mouse Leydig cell line TM3 was stimulated with H2O2. ELISA was employed for testosterone level evaluation. Hematoxylin-eosin staining was implemented for histologic analysis of mouse testicular tissues. Western blotting and RT-qPCR were utilized for evaluating molecular protein and RNA expression, respectively. Functional experiments were conducted to test miR-361-5p roles. Luciferase reporter assay was for verifying the interaction between miR-361-5p and protein inhibitor of activated STAT 1 (PIAS1). miR-361-5p displayed a decreased level in the testes of LOH mice. Overexpressing miR-361-5p attenuated Leydig cell loss in the testis and elevated serum and intratesticular testosterone levels in LOH mice. H2O2 stimulation impaired TM3 cell viability, proliferation and intracellular testosterone production and enhanced cell apoptosis. miR-361-5p targeted PIAS1 in TM3 cell. PIAS1 upregulation counteracted miR-361-5p overexpression-mediated alleviation of cell apoptosis and elevation of testosterone synthesis in H2O2-stimualetd TM3 cells. miR-361-5p ameliorates LOH progression by increasing testosterone production and alleviate Leydig cell apoptosis via downregulation of PIAS1.
摘要：
迟发性性腺功能减退症（LOH）是一种与年龄有关的综合征，其特征是Leydig细胞产生的血清睾丸激素缺乏。先前的证据表明，microRNA（miR）-361-3p可以作为LOH的有希望的生物标志物。尽管如此，其在LOH中的详细功能和分子机制尚不清楚。选择24月龄雄性小鼠作为LOH动物模型,用H2O2刺激小鼠Leydig细胞系TM3。采用ELISA评价睾酮水平。苏木精-伊红染色用于小鼠睾丸组织的组织学分析。蛋白质印迹和RT-qPCR用于评估分子蛋白和RNA表达。分别。进行功能实验以测试miR-361-5p的作用。荧光素酶报告基因测定用于验证miR-361-5p与活化的STAT1(PIAS1)的蛋白抑制剂之间的相互作用。miR-361-5p在LOH小鼠睾丸中显示出降低的水平。过表达miR-361-5p减弱了LOH小鼠睾丸间质细胞损失和血清和睾丸内睾酮水平升高。H2O2刺激损害TM3细胞活力，增殖和细胞内睾酮产生和增强细胞凋亡。miR-361-5p靶向TM3细胞中的PIAS1。PIAS1上调抵消了miR-361-5p过表达介导的H2O2刺激TM3细胞凋亡和睾酮合成升高的缓解。miR-361-5p通过增加睾酮产生改善LOH进展，并通过下调PIAS1减轻Leydig细胞凋亡。
