ABSTRACT: Abstract Background: The natural compound paeonol exhibits therapeutic promise against cervical carcinoma, though its precise molecular mechanisms remain undefined. Methods: First, we treated human cervical cancer (HeLa) cells with different concentrations of paeonol. Cellular proliferation and apoptotic responses were evaluated via cell-counting kit 8 (CCK8) assays and flow cytometric analysis. Subsequent transcriptomic profiling employed RNA sequencing coupled with alternative splicing assessment to detect differentially expressed genes (DEGs). Protein interaction networks were established for pivotal DEGs, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment investigations. Clinical data pertinent to cervical cancer were retrieved from The Cancer Genome Atlas (TCGA). Prognostic model development incorporated Kaplan–Meier survival estimation, Least Absolute Shrinkage and Selection Operator (LASSO) regression, alongside univariate and multivariate COX proportional hazards analyses, with model accuracy subsequently assessed. Finally, Quantitative reverse transcription polymerase chain reaction (qRT-PCR) validated DEG expression. Results: Paeonol treatment suppressed proliferation while inducing apoptosis in HeLa cells. Transcriptomic and splicing analyses revealed 12 critical DEGs: NLRP1, FN1, NQO2, NREP, B4GALNT1, ANK3, FAM219A, ODF3B, MAPK15, EPGN, MUC1, and MEG3. Enrichment analyses indicated these DEGs principally associate with inflammatory processes and the biological regulation of cellular proliferation and apoptotic death. Analysis of clinical outcomes in 197 TCGA patients demonstrated significantly enhanced five-year survival probability within the low-risk cohort. FN1, NQO2, and ODF3B were incorporated into a prognostic signature following LASSO regression. Univariate and multivariate COX analyses identified T stage, tumor grade, and differential expression of these three genes as significant outcome predictors; the resultant prognostic model exhibited robust accuracy. qRT-PCR results corroborated the RNA sequencing data concerning DEG expression patterns. Conclusion: Paeonol modulates HeLa cell proliferation and apoptosis through regulation of 12 key genes, including FN1. This activity involves governing inflammatory responses alongside cellular proliferation, migration, and differentiation processes. These findings offer a theoretical foundation supporting paeonol's potential clinical utility in cervical cancer management.
