Role And Mechanism Of Plk1 In Phosphoramide Mustard-induced Ovarian Granulosa Cell Injur

Objective: The molecular mechanisms underlying the damage to ovarian granulosa cells(GCs)caused by Phosphoramidemustard(PM)are complex and not well understood,and the specific genes and signaling pathways involved remain unclear.This study aimed to investigate the key genes and potential mechanisms of PM-induced damage to GCs using RNA-seq technology and bioinformatics analysis.In addition,we conducted in vitro experiments to explore the regulatory effects of key genes on cell function and downstream signaling pathways.Methods: 1.In vitro experiments were conducted to evaluate cell viability and apoptosis rate in GCs following PM-induced injury,with normal GCs used as controls.RNA-seq and bioinformatics analysis were performed on the sequencing data,and differentially expressed genes were subjected to GO and KEGG pathway enrichment analyses.Gene Set Enrichment Analysis(GSEA)was also conducted to identify hub genes and determine the potential mechanisms of damage.2.Overexpression of Polo-like Kinase 1(Plk1),a key gene,was performed in GCs.The experimental groups included the control group(Control),PM group(PM),overexpression empty vector group(OE-NC),overexpression empty vector + PM group(OE-NC+PM),overexpression Plk1 group(OE-Plk1),and overexpression Plk1 + PM group(OE-Plk1+PM).Cell viability,Ed U staining,and flow cytometry were used to assess apoptosis and cell cycle changes in GCs after overexpression.3.q RT-PCR and Western blotting were employed to measure the mRNA and protein levels of Bax,Bcl-2,Caspase-3,and p-AKT.Results: 1.The sequencing results revealed 1261 differentially expressed genes in the PM group compared to the normal group,with 715 upregulated and 546 downregulated genes.GO enrichment analysis of the differentially expressed genes showed enrichment in biological processes related to chromosome segregation,nuclear division,and organelle division.The cellular component analysis indicated enrichment in condensed chromosomes,chromosomal trophoblast region,and chromosomal regions.The molecular function analysis revealed enrichment in catalytic activity,receptor ligand activity,and catalytic activity related to DNA.KEGG pathway analysis demonstrated that downregulated genes were involved in DNA replication,cell cycle regulation,and the interleukin-17 signaling pathway,while upregulated genes were associated with the MAPK signaling pathway,cytochrome P450 metabolism of exogenous substances,and the chemokine-cytokine signaling pathway.The GSEA enrichment analysis of gene sets included the cell cycle,interleukin-17 signaling pathway,and tumor necrosis factor signaling pathway.2.Compared to the normal group,the PM group exhibited decreased cell viability,reduced cell proliferation,increased apoptosis,and disrupted cell cycle(p < 0.05).The OE-NC+PM group showed similar changes compared to the OE-NC group,while the OE-Plk1+PM group demonstrated increased cell viability,enhanced cell proliferation,reduced apoptosis,and improved cell cycle compared to the PM group and OE-NC+PM group(p< 0.05).3.Compared to the normal group,the PM group displayed increased mRNA levels of Bax and Caspase-3,elevated protein levels of Bax and Cleaved-Caspase-3,decreased mRNA and protein levels of Bcl-2,and decreased p-AKT protein level(p < 0.05).In the OE-Plk1+PM group,the mRNA levels of Bax and Caspase-3 decreased,as did the protein levels of Bax and Cleaved-Caspase-3,while the mRNA and protein levels of Bcl-2 increased,and the p-AKT protein level increased(p < 0.05).Conclusions: 1.RNA-seq technology and bioinformatics analysis identified multiple genes and signaling pathways involved in PM-induced damage to GCs,with Plk1 identified as a key gene.2.Overexpression of Plk1 suppressed apoptosis and promoted cell proliferation in PM-injured GCs.3.Overexpression of Plk1 regulated the AKT pathway,upregulated Bcl-2,and downregulated Bax and Caspase-3,thereby inhibiting PM-induced apoptosis in GCs.