Integrated bioinformatics, machine learning, and experimental validation identify a four-gene diagnostic signature...

Early and accurate diagnosis remains a major challenge in cervical cancer management. This study aimed to identify reliable diagnostic biomarkers for cervical cancer by integrating bioinformatics and machine learning approaches and to further validate their biological relevance experimentally. Transcriptomic data from the Gene Expression Omnibus and The Cancer Genome Atlas were analyzed using differential expression analysis, weighted gene co-expression network analysis, and three machine learning algorithms to identify core genes. Diagnostic performance was evaluated using receiver operating characteristic curves and a nomogram model. Functional relevance was explored by drug sensitivity analysis, ssGSEA, immune infiltration analysis, and single-cell RNA sequencing. RT-qPCR validation was performed in 10 paired cervical cancer and adjacent normal tissues, while Western blotting was performed in three paired tissue samples. In vitro validation was conducted using SiHa and HeLa cells. Four genes, CCND1, TRIP13, MYBL2, and GNB4, were identified as potential diagnostic biomarkers, and the combined model showed superior diagnostic performance compared with any single gene (AUC = 0.989). Treatment with 3-methyladenine altered the expression of these genes, suggesting their potential association with PI3K/AKT-related pathway activity. Moreover, siRNA-mediated GNB4 knockdown suppressed cervical cancer cell proliferation and reduced PI3K and AKT phosphorylation, providing preliminary evidence for the functional involvement of GNB4 in PI3K/AKT pathway activation. CCND1, TRIP13, MYBL2, and GNB4 may serve as promising diagnostic biomarkers for cervical cancer. Their dysregulation was associated with PI3K/AKT pathway activity and may reflect molecular alterations involved in cervical cancer progression. In particular, GNB4 showed potential diagnostic relevance and preliminary functional significance, suggesting that it may represent a candidate biomarker and molecular target for further investigation.