Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Exploitation of Ministry of Education, Southwest Minzu University, Chengdu, Sichuan, 610041, PR China.
Key Laboratory for Animal Science of National Ethnic Affairs Commission, Southwest Minzu University, Chengdu, Sichuan, 610041, PR China.
Theriogenology. 2022 Dec;194:46-57. doi: 10.1016/j.theriogenology.2022.09.026. Epub 2022 Sep 29.
Cattleyaks, a hybrid of (♂) and yak (♀), exhibit the marked productivity and adaptability of plateau, but suffer from male infertility. Small non-coding RNAs, especially miRNAs, play crucial roles in spermatogenesis and affect the growth of Sertoli cells (SCs). The objective of the present study was to explore the interaction between miR-34b-5p and protein phosphatase 1 regulatory inhibitor subunit 11 (PPP1R11) and its effect on cattleyak SCs. RT-qPCR was used to determine the expression pattern of miR-34b-5p and PPP1R11, while the cellular and subcellular localization of PPP1R11 was determined by immunohistochemistry and immunocytochemistry. The interaction between MiR-34b-5p and PPP1R11 was evaluated by immunofluorescence, proliferation, apoptosis, and western blotting assays. The potential binding sites between miR-34b-5p and PPP1R11 were uncovered through targeted search of an online database, and verified using a dual luciferase reporter system. Our data show that miR-34b-5p is differentially expressed in the testes and SCs of cattleyaks compared to yaks. Overexpression of miR-34b-5p in SCs suppressed proliferation and induced apoptosis, while the effects of miR-34b-5p knockdown were the reverse. The 3'UTR of PPP1R11 was identified as a potential target site of miR-34b-5p, and this was validated by online database searches and our data from the dual-luciferase reporter assay, and it displayed an inverse expression pattern to miR-34b-5p in SCs. The effects of silencing PPP1R11 by siRNA were similar to the results of miR-34b-5p upregulation, but significantly different from miR-34b-5p downregulation in cattleyak SCs. The effects with PPP1R11 overexpression were opposite, suggesting a novel biofunctional role of PPP1R11 inactivation in depressing cattleyak SCs growth. Lastly, we confirmed that miR-34b-5p inhibited PPP1R11 expression and induced apoptosis by regulating proliferation- and apoptosis-related genes in SCs. Thus, miR-34b-5p regulates the apoptosis and proliferation of cattleyak SCs via targeting PPP1R11, which can provide an innovative direction for exploring the mechanism of cattleyak male sterility.
考特利亚克羊是（♂）和牦牛（♀）的杂交种，具有高原地区的显著生产力和适应性，但存在雄性不育问题。小非编码 RNA，特别是 microRNA，在精子发生中发挥关键作用，并影响支持细胞（SCs）的生长。本研究旨在探讨 miR-34b-5p 与蛋白磷酸酶 1 调节抑制剂亚基 11（PPP1R11）之间的相互作用及其对考特利亚克 SCs 的影响。通过 RT-qPCR 确定 miR-34b-5p 和 PPP1R11 的表达模式，通过免疫组织化学和免疫细胞化学确定 PPP1R11 的细胞和亚细胞定位。通过免疫荧光、增殖、凋亡和 Western blot 测定评估 MiR-34b-5p 和 PPP1R11 之间的相互作用。通过在线数据库的靶向搜索发现 miR-34b-5p 与 PPP1R11 之间的潜在结合位点，并通过双荧光素酶报告系统进行验证。我们的数据表明，miR-34b-5p 在考特利亚克羊的睾丸和 SCs 中的表达与牦牛不同。在 SCs 中过表达 miR-34b-5p 抑制增殖并诱导细胞凋亡，而 miR-34b-5p 敲低的作用则相反。PPP1R11 的 3'UTR 被鉴定为 miR-34b-5p 的潜在靶标位点，这通过在线数据库搜索和我们的双荧光素酶报告实验数据得到验证，并且在 SCs 中与 miR-34b-5p 的表达呈负相关。通过 siRNA 沉默 PPP1R11 的效果与 miR-34b-5p 上调的结果相似，但与考特利亚克羊 SCs 中 miR-34b-5p 下调的结果明显不同。PPP1R11 过表达的效果则相反，这表明 PPP1R11 失活在抑制考特利亚克羊 SCs 生长方面具有新的生物功能作用。最后，我们证实 miR-34b-5p 通过调节 SCs 中的增殖和凋亡相关基因来抑制 PPP1R11 的表达并诱导细胞凋亡。因此，miR-34b-5p 通过靶向 PPP1R11 调节考特利亚克羊 SCs 的凋亡和增殖，为探索考特利亚克羊雄性不育机制提供了新的思路。
