基于生物信息学与功能实验探究RFC5在卵巢癌复发及干性维持中的作用OA

Objective To identify the recurrence-and stemness-associated genes,particularly replication factor C subunit 5(RFC5)in ovarian cancer through bioinformatic analysis and experimental validation,and to elucidate its underlying molecular mechanisms.Methods Primary ovarian cancer cells were isolated from tumor tissues obtained from 7 patients with ovarian cancer who were treated at the Department of Obstetrics and Gynecology,West China Second University Hospital,Sichuan University,between March 2024 and March 2025.Ovarian cancer stem cells(OCSCs)were enriched through serum-free suspension culture and subjected to transcriptomic sequencing.Weighted gene co-expression network analysis(WGCNA)was performed to identify key recurrence and stemness-associated candidate genes.The Kaplan-Meier Plotter database was used to analyze the clinical prognostic value.The CIBERSORT algorithm was employed to assess the association between RFC5 and immune microenvironment.Based on single-cell transcriptomic data,pseudotime trajectory and in silico knockdown analyses were conducted to investigate the role of RFC5 in tumor recurrence and the associated downstream pathways.RFC5-knockdown and RFC5-overexpression OCSC models were separately constructed via siRNA interference and lentiviral infection.Quantitative reverse transcription polymerase chain reaction(qRT-PCR)was used to detect the RFC5 expression in OCSCs and differentiated ovarian cancer cells(Diff-OCs).Sphere formation assay and CCK-8 assays were used to evaluate the effects of RFC5 on OCSCs sphere-forming capacity and proliferation activity.Results Seven candidate genes(ZNF157,SULT2B1,ELL3,DMRT3,SAPCD2,RFC5,ZBTB9)were identified as key recurrence and stemness-associated genes.Among them,RFC5 was significantly upregulated in ovarian cancer tissues compared with normal tissues(P<0.001),and high RFC5 expression was significantly associated with shorter progression-free survival(PFS,HR=1.23,95%CI:1.08-1.41,P=0.002)and overall survival(OS,HR=1.17,95%CI:1.01-1.36,P=0.039).RFC5 expression showed a significant negative correlation with naïve CD4+T-cell infiltration(r=-0.745)and positive correlations with multiple immune checkpoint molecules including CD276(r=0.662),TNFRSF9(r=0.514),and LGALS9(r=0.552).Single-cell pseudotime analysis demonstrated that RFC5 was progressively upregulated of along the trajectory from stem-like cells to recurrent tumor cells.Following in silico RFC5 knockdown,genes associated with ribosomal proteins,metabolic molecules,and tumor immune regulation were significantly altered and enriched in oxidative phosphorylation,thermogenesis,and ROS-related carcinogenesis pathways.In vitro experiments confirmed that RFC5 was highly expressed in OCSCs(P<0.05);knockdown of RFC5 significantly inhibited sphere-forming capacity and cell proliferation,while overexpression of RFC5 produced opposite effects(all P<0.05).Conclusions RFC5 serves as a key recurrence-and stemness-associated gene in ovarian cancer promotes sphere formation and proliferation of OCSCs.It may contribute to tumor recurrence through modulation of the immune microenvironment and regulating metabolic pathways such as oxidative phosphorylation.RFC5 represents a potential prognostic biomarker and therapeutic target for anti-recurrence treatment in ovarian cancer.However,further validation in larger cohorts and detailed mechanistic studies are still warranted.