Objective: Being a prevalent endocrine and metabolic disease, polycystic ovary syndrome (PCOS) severely threatens women's physical and mental health. Glioma-associated oncogene family zinc finger 2 (GLI2) expression is up-regulated in granulosa cells of PCOS patients, but its specific role in PCOS remains unclear.
Methods: Following the treatment of human ovarian granulosa cells (KGN) with dihydrotestosterone (DHT), RT-qPCR and western blot were utilized to check GLI2 expression. After GLI2 expression was silenced, cell activity was detected through CCK8 and apoptosis was examined via TUNEL and western blot. Inflammation and oxidative stress were tested utilizing ELISA and western blot. The binding between GLI2 and neuronal precursor cell-expressed developmentally downregulated 4 (NEDD4L) promoter was predicted by JASPAR database and verified by luciferase reporter and ChIP assay. In addition, RT-qPCR and western blot were applied to check the mRNA and protein expressions of NEDD4L. Following the knockdown of NEDD4L in GLI2-silencing cells, CCK8 assay, TUNEL assay, western blot, ELISA and other methods were performed again. Finally, western blot detected the expressions of Wnt pathway-related proteins.
Results: GLI2 was up-regulated in DHT-treated KGN cells. Interference with GLI2 increased the viability, decreased the apoptosis, and inhibited the inflammatory response and oxidative stress of DHT-induced KGN cells. GLI2 could bind to NEDD4L promoter and transcriptionally suppress NEDD4L expression. Further experiments testified that NEDD4L depletion reversed the impacts of GLI2 deficiency on the viability, apoptosis, inflammation, oxidative stress and Wnt signaling pathway in DHT-challenged KGN cells.
Conclusion: GLI2 activated Wnt signaling to promote androgen-induced granulosa cell damage through transcriptional inhibition of NEDD4L.
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