Hunan Provincial Key Laboratory for Genetic Improvement of Domestic Animal, College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, China.
Yi Chuan. 2021 Jul 20;43(7):680-693. doi: 10.16288/j.yczz.21-154.
The number of Sertoli cells in the testis is a major regulator on the sperm production capacity. MicroRNAs (miRNAs) participate in regulating the proliferation and apoptosis of porcine immature Sertoli cells. However, the functions and mechanisms of action of most identified miRNAs in porcine Sertoli cells remain largely unknown. In the present study, based on our previous results from an EdU-based high-content screening assay, we further studied the mechanism of action of miR-191 on the proliferation and apoptosis of porcine immature Sertoli cells through flow cytometry, Western blotting, and dual-luciferase activity analyses. The results demonstrated that overexpression of miR-191 promoted cell cycle progression from G phase to the S and G phases, enhanced cell proliferation, and inhibited apoptosis in the porcine immature Sertoli cells, whereasmiR-191 inhibition resulted in the opposite effects. The results from a luciferase reporter assay showed that miR-191 directly targeted the 3'-UTR of theBDNF gene. BDNF knockdown also promoted cell cycle progression to the S phase, cell proliferation and inhibited cell apoptosis, which were consistent with the effects of the miR-191overexpression. A co-transfection experiment showed that BDNF knockdown abolished the effects of miR-191 inhibition. Furthermore, both miR-191 overexpression and BDNFinhibition elevated the phosphorylation of PI3K and AKT, the key components of the PI3K/AKT signaling pathway, whereas BDNFinhibition offset the effects of the miR-191 knockdown. Overall, these data indicated that miR-191 promotes cell proliferation and inhibits apoptosis in porcine immature Sertoli cells by targeting theBDNF gene through activating the PI3K/AKT signaling pathway. This study provides a novel scientific basis for further investigation on the biological functions of miR-191 on porcine spermatogenesis.
睾丸中的支持细胞数量是精子产生能力的主要调节因子。微小 RNA（miRNA）参与调节猪未成熟支持细胞的增殖和凋亡。然而，大多数已鉴定的 miRNA 在猪支持细胞中的功能和作用机制仍知之甚少。在本研究中，基于我们之前基于 EdU 的高通量筛选试验的结果，我们通过流式细胞术、Western blot 分析和双荧光素酶活性分析进一步研究了 miR-191 对猪未成熟支持细胞增殖和凋亡的作用机制。结果表明，miR-191 的过表达促进了细胞周期从 G 期向 S 和 G 期的进展，增强了猪未成熟支持细胞的增殖，抑制了细胞凋亡，而 miR-191 的抑制则产生了相反的效果。荧光素酶报告基因检测结果表明，miR-191 直接靶向 BDNF 基因的 3'-UTR。BDNF 敲低也促进了细胞周期向 S 期的进展、细胞增殖并抑制细胞凋亡，这与 miR-191 过表达的效果一致。共转染实验表明，BDNF 敲低消除了 miR-191 抑制的作用。此外，miR-191 过表达和 BDNF 抑制均提高了 PI3K/AKT 信号通路的关键组成部分 PI3K 和 AKT 的磷酸化水平，而 BDNF 抑制则抵消了 miR-191 敲低的作用。总体而言，这些数据表明，miR-191 通过激活 PI3K/AKT 信号通路靶向 BDNF 基因促进猪未成熟支持细胞的增殖并抑制凋亡。本研究为进一步研究 miR-191 在猪精子发生中的生物学功能提供了新的科学依据。
