Table_3_miR-205 Expression Elevated With EDS Treatment and Induced Leydig Cell Apoptosis by Targeting RAP2B via the PI3K/AKT Signaling Pathway.XLSX

The adult Leydig cells (ALCs), originated from stem Leydig cells (SLCs), can secrete testosterone which is essential for germ cell development and sexual behavior maintenance. As a synthetic compound, ethane dimethane sulfonate (EDS), a well-known alkylating agent, has been reported to specifically ablate ALCs. In this study, EDS was verified to ablate differentiated pig LCs by experiments. Subsequently, the primary isolated pig LCs (containing SLCs and differentiated LCs) and EDS-treated LCs (almost exclusively SLCs) were collected for RNA-seq 4,904 genes and 15 miRNAs were differently expressed between the two groups. Down-regulated genes in the EDS-treated group were mainly related to steroid hormone biosynthesis. The highest up-regulation miRNAs was miR-205 after EDS treatment. Additionally, miR-205 was expressed more highly in pig SLCs clones compared with differentiated LCs. Through qRT-PCR, western blot (WB), TUNEL, EDU and flow cytometry, miR-205 was found to induce cell apoptosis, but did not affect proliferation or differentiation in both TM3 and GC-1spg mouse cell lines. Through luciferase reporter assays and WB, RAP2B was identified as a target gene of miR-205. Besides, overexpression of miR-205 inhibited the expressions of PI3K, Akt and p-AKT. All these findings were helpful for elucidating the regulation mechanism in pig LCs.

成年睾丸间质细胞（adult Leydig cells, ALCs）由干细胞样睾丸间质细胞（stem Leydig cells, SLCs）分化而来，可分泌睾酮，而睾酮对生殖细胞发育及性行为维持至关重要。作为一种合成化合物，乙烷二甲基磺酸盐（ethane dimethane sulfonate, EDS）是经典的烷化剂，据报道可特异性清除ALCs。本研究通过实验证实，EDS可清除分化状态的猪睾丸间质细胞（pig LCs）。随后，研究人员收集原代分离的猪睾丸间质细胞（包含SLCs与分化型LCs）以及经EDS处理后的睾丸间质细胞（几乎仅存SLCs）进行RNA-seq检测，两组间共鉴定出4904个差异表达基因与15个差异表达miRNA。EDS处理组的下调基因主要富集于类固醇激素生物合成通路。EDS处理后上调幅度最高的miRNA为miR-205。此外，与分化型LCs相比，猪SLCs克隆中的miR-205表达水平更高。通过qRT-PCR、蛋白质印迹（western blot, WB）、TUNEL、EDU及流式细胞术实验，研究发现miR-205可诱导TM3与GC-1spg小鼠细胞系发生细胞凋亡，但不影响细胞增殖与分化。通过荧光素酶报告基因实验与蛋白质印迹实验，证实RAP2B是miR-205的靶基因。此外，过表达miR-205可抑制PI3K、Akt及p-AKT的表达。上述研究结果有助于阐明猪睾丸间质细胞的调控机制。