Study of differential proteomics in granulosa cells of premature ovarian insufficiency (POI) and the roles and mechanism of RAC1 in granulosa cells

In the present study, we focused on characterizing the proteome in granulosa cells in patients with biochemical premature ovarian insufficiency (bPOI) in order to identify differential proteins and investigate the fundamental mechanisms of POI. A total of 2688 proteins were identified based on the data-independent acquisition method, and 70 differentially expressed proteins were significant. Bioinformatic analyses, including gene expression pattern analysis, gene ontology enrichment analysis, Kyoto Encyclopedia of Genes and Genomes pathway analysis, and Search Tool for the Retrieval of Interacting Genes/Proteins analysis, revealed discrete modules and the underlying molecular mechanisms in bPOI. Importantly, we observed that Ras-related C3 botulinum toxin substrate 1 (RAC1) was downregulated in the granulosa cells of bPOI. Low expression of RAC1 may affect the development process of POI by affecting the proliferation, Apoptosis, and hormone synthesis of granulosa cells. Downregulation of RAC1 expression in the KGN and COV434 cells inhibited cell proliferation, blocked cells in the G1/G0 phase, and promoted Apoptosis. Western blot results showed that β-catenin and cyclin D1 in the KGN and COV434 cells transfected with RAC1-siRNA were downregulated, while P21 and Bax were upregulated. Knocking down RAC1 in the KGN cells or adding the RAC1 enzyme inhibitor to the human luteinized granulosa cells (hLGC) inhibited the synthesis of E₂, and the expression of aromatase and follicle-stimulating hormone receptor (FSHR) was reduced.