ABSTRACT: Background: Lidocaine, a widely used local anesthetic, has been reported to exert anti-cancer activity against hepatocellular carcinoma (HCC). However, its molecular mechanisms remain incompletely understood. This study sought to elucidate the mechanisms underlying lidocaine’s effects on HCC. Methods: Potential lidocaine targets in HepG2 cells were identified using network pharmacology and transcriptomic profiling. The prognostic and clinical relevance of candidate genes were assessed through bioinformatics analyses. Key targets were validated by RT–qPCR. The functional role of SLC6A3 in regulating HepG2 cell proliferation, apoptosis, migration, and invasion was examined through in vitro assays. Results: Network pharmacology predicted 433 lidocaine targets, while transcriptomic profiling revealed 442 differentially expressed genes. Nine overlapping targets (SLC6A3, CHRNB2, GRIN1, ADRA2C, LIPE, SLC18A2, KCNQ2, TERT, and ALOX12) were enriched in pathways associated with neuronal signaling, synaptic transmission, and drug addiction. Among these, SLC6A3 and TERT were significantly associated with poor prognosis and increased tumor immune infiltration. Both genes demonstrated predictive value for 1- to 2-year survival, with SLC6A3 showing the stronger prognostic relevance. Molecular docking revealed hydrophobic interactions between lidocaine and SLC6A3 (binding energy: −5.6 kcal/mol). Silencing of SLC6A3 markedly promoted apoptosis and suppressed proliferation, migration, and invasion of HepG2 cells. Collectively, these findings suggest that lidocaine inhibits HCC progression by targeting and downregulating SLC6A3. Conclusion: Lidocaine exerts anti-HCC effects by directly targeting and downregulating SLC6A3, thereby inducing apoptosis and suppressing tumor progression.
