Dai Tianshu, Wei Shihao, Li Xiaofeng, Yang Di, Gui Linsen, Xiang Hui, Ma Yun, Dan Xingang
College of Animal Science and Technology, Ningxia University, Yinchuan 750021, China.
College of Animal Science, Anhui Science and Technology University, Fengyang 233100, China.
Domest Anim Endocrinol. 2025 Jul;92:106947. doi: 10.1016/j.domaniend.2025.106947. Epub 2025 May 9.
The aim of this study was to verify the hypothesis that kisspeptin, a peptide encoded by the kiss1 gene, regulates steroidogenesis and cell proliferation in ovarian granulosa cells (GCs) from Tan sheep through modulation of let-7b and ITGB7 (integrin subunit beta 7). First, primary ovarian GCs were transfected with let-7b mimics and inhibitors. Next, HEK293T cells were cultured to validate the targeting relationship between let-7b and ITGB7, followed by the overexpression and knockdown of ITGB7 in GCs. Finally, GCs were treated with the PI3K-AKT/ERK signaling pathway inhibitor and 500 nM kisspeptin after transfection with ITGB7. EdU assays, flow cytometry, quantitative PCR (qPCR) and Western blotting were then used to detect cell proliferation, cell cycle and apoptosis as well as related gene and protein expression. The results showed that let-7b significantly inhibited progesterone secretion and cell proliferation while promoting apoptosis in GCs by targeting ITGB7. Notably, overexpression of ITGB7 led to a marked upregulation of p-ERK/ERK, p-PI3K/PI3K, and p-Akt/Akt. Furthermore, co-treatment with kisspeptin and ITGB7 significantly enhanced progesterone secretion and cell proliferation while reducing apoptosis in ovarian GCs. These results provide novel insights into the mechanism by which kisspeptin downregulates let-7b and upregulates ITGB7, thereby promoting steroidogenesis and cell proliferation while inhibiting apoptosis via the ERK/PI3K-Akt signaling pathway in Tan sheep. This study provides new insights into the molecular mechanisms by which kisspeptin regulates the function of ovarian GCs, and may lay the foundation for the future development of new kisspeptin-mediated reproductive regulation techniques in Tan sheep.
由kiss1基因编码的一种肽——亲吻素，通过调节let-7b和整合素β7亚基（ITGB7）来调控滩羊卵巢颗粒细胞（GCs）中的类固醇生成和细胞增殖。首先，用let-7b模拟物和抑制剂转染原代卵巢颗粒细胞。接下来，培养人胚肾293T细胞以验证let-7b与ITGB7之间的靶向关系，随后在颗粒细胞中过表达和敲低ITGB7。最后，在用ITGB7转染颗粒细胞后，用PI3K-AKT/ERK信号通路抑制剂和500 nM亲吻素处理颗粒细胞。然后使用EdU检测、流式细胞术、定量聚合酶链反应（qPCR）和蛋白质免疫印迹法来检测细胞增殖、细胞周期和凋亡以及相关基因和蛋白质表达。结果表明，let-7b通过靶向ITGB7显著抑制颗粒细胞中的孕酮分泌和细胞增殖，同时促进细胞凋亡。值得注意的是，ITGB7的过表达导致p-ERK/ERK、p-PI3K/PI3K和p-Akt/Akt显著上调。此外，亲吻素与ITGB7联合处理显著增强了卵巢颗粒细胞中的孕酮分泌和细胞增殖，同时减少了细胞凋亡。这些结果为亲吻素下调let-7b并上调ITGB7的机制提供了新的见解，从而通过ERK/PI3K-Akt信号通路促进类固醇生成和细胞增殖，同时抑制滩羊颗粒细胞凋亡。本研究为亲吻素调节卵巢颗粒细胞功能的分子机制提供了新的见解，并可能为未来开发新的亲吻素介导的滩羊生殖调控技术奠定基础。
