A novel mechanism of kisspeptin regulating ovarian granulosa cell function via down-regulating let-7b to activate ERK/PI3K-Akt pathway in Tan sheep

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.