鼻咽癌增殖转录程序的跨队列鉴定及eIF4A相关翻译调控的定位:274个基因增殖候选集构建与Silvestrol体外验证OA

Objective Nasopharyngeal carcinoma(NPC)is characterized by marked geographic aggregation,Epstein-Barr virus(EBV)association,and biological heterogeneity;local recurrence,distant metastasis,and acquired resistance remain major obstacles to long-term benefit,while single-gene differential expression is vulnerable to cohort and platform effects.This study aims to identify transcriptional programs in NPC with cross-cohort reproducibility and relevance to disease progression,construct a proliferation candidate gene set supported by independent expression data,and evaluate its relationship with eukaryotic translation initiation factor 4A(eIF4A)-associated translational regulation and the in vitro pharmacological effects of Silvestrol.Methods ① Bioinformatic analyses of public datasets:Public transcriptomic datasets(GSE12452,GSE53819,GSE102349,GSE68799,and GSE150430)were integrated.Unbiased gene set enrichment analysis was performed in GSE12452 and GSE53819 to identify pathways showing concordant enrichment directions and a false discovery rate(FDR)<0.05 in both cohorts.Weighted gene co-expression network analysis was conducted in GSE102349,and stage-stratified Cox models were applied to explore the associations between module eigengenes and disease progression.A 274-gene proliferation candidate set was constructed by integrating cross-cohort differential expression with module membership,and Top50,Top100,and Top150 nested gene sets were then used for Library of Integrated Network-based Cellular Signatures(LINCS)connectivity and mechanism-of-action analyses.Within-sample associations between translation-related programs and eIF4A-related transcripts were further evaluated and externally assessed in GSE68799.EIF4A1 functional loss was simulated in malignant cells using scTenifoldKnk to characterize network perturbation patterns.2 In vitro experiments:CCK-8,cell-killing,colony formation,and wound-healing assays were used to determine the effects of Silvestrol on cultured 5-8F and CNE-2Z cells.Results The normalized enrichment score(NES)of 5 124 commonly evaluable pathways exhibited a positive Spearman-rank correlation between the GSE12452 and GSE53819 cohorts(Spearman ρ,P<2.2×10-16).A total of 314 concordantly activated and 21 concordantly suppressed pathways were identified,characterized mainly by enhanced cell cycle,DNA replication,and chromosome regulation programs and reduced cilium-and axoneme-related processes,respectively.Fifteen non-grey modules were identified in GSE102349.Among 68 patients with complete clinical data,14 disease progression events were recorded,and the Turquoise,Tan,Blue,and Pink modules reached nominal P<0.05.Integration of Turquoise-module membership with concordant upregulation in the 2 external cohorts yielded 274 proliferation candidate genes with cross-cohort expression support,which were enriched mainly in E2F,G2/M,MYC,DNA metabolism,and chromosome segregation programs.In the LINCS analysis,connectivity rankings for 8 140 compounds were highly correlated(r=0.909 to 0.955).Protein-synthesis inhibitors showed significant negative connectivity in the Top50 and Top100 analyses,with FDR values of 0.016 and 0.040,respectively.Translation program analysis indicated process-selective alterations in NPC rather than coordinated activation of the entire translation system.In the external GSE68799 cohort,the proliferation-adjusted correlations of EIF4B with general translation initiation and of PDCD4 with mRNA surveillance/NMD were r=0.510 and 0.505,respectively,with FDR=0.012 for both;associations between canonical EIF4A1 and selected programs were model-sensitive.Single-cell analysis of GSE150430 showed that EIF4A1 was detectable in 95.81%of malignant cells.Virtual perturbation signals were concentrated mainly in RPL/RPS ribosomal-protein genes and mitochondrially encoded transcripts,whereas the complete proliferation candidate set was not enriched at the high-perturbation end of the ranking(FDR=1.000).The half-maximal inhibitory concentrations of Silvestrol were 26 and 12 nmol/L in 5-8F and CNE-2Z cells,respectively,and Silvestrol reduced cell retention,colony formation,and wound closure.Conclusion NPC exhibits a cross-cohort reproducible transcriptional state characterized by enhanced proliferative programs,with eIF4A-associated translational regulation prioritized by multi-level analyses as a candidate intervention mechanism;Silvestrol exhibits nanomolar in vitro inhibitory activity in NPC cells.