RNA Sequencing Explores Potential Mechanisms Underlying the Enhanced Aggressive Phenotype of HepG2 Cells Induced by Aged Zinc Oxide Nanoparticles

Purpose: While aged zinc oxide nanoparticles (ZnO NPs) show reduced acute cytotoxicity, their chronic effects remain unclear. This study compared the long-term, low-dose impact of environmentally aged versus fresh ZnO NPs on enhanced aggressive phenotype in hepatocellular carcinoma cells. Material and Methods: HepG2 cells were chronically exposed (approximately 16 weeks) to low doses (1.5 μg/mL) of aged or fresh ZnO NPs. RNA sequencing (n=3 per group) identified transcriptomic changes, while migration and invasion assays validated functional outcomes. A transcription factor (TF) -mRNA network was constructed, and clinical correlation was analyzed using patient survival data. Results: Aged ZnO NPs were associated with the activation of pro-oncogenic pathways (e.g. JAK-STAT) and induced increased cell migration and invasion compared to fresh NPs. Network analysis suggested CEBPA, CTNNB1, and STAT3 as potential core transcriptional regulators. Consistent with their potential role in promoting an aggressive phenotype, the elevated expression levels of these TFs were associated with reduced overall survival in patients with hepatocellular carcinoma. Conclusion: Chronic exposure to environmentally aged ZnO NPs, despite lower acute toxicity, may promote a more aggressive phenotype in liver cells compared to fresh NPs. This effect is potentially mediated by the specific modulation of oncogenic pathways and a core transcriptional network linked to poor patient outcomes. Our findings suggest that the environmental aging process could be an important factor influencing the long-term potential of ZnO NPs to promote an aggressive phenotype, which should be considered in nanomaterial risk assessment. The diagram illustrates a process flow starting with aging and cell culture. It shows control, fresh nanoparticles and aged nanoparticles, followed by characterization using TEM, DLS and ICP-OES. Next is RNA sequencing, enrichment and the TF-mRNA network. Functional validation is depicted with a container and particles. Finally, clinical correlation is shown with a graph of survival probability over time, comparing high and low expression levels.Diagram showing aging and cell culture, RNA sequencing, functional validation and clinical correlation processes. Keywords: zinc oxide nanoparticles, aging, HepG2 cells, transcriptomics, enhanced aggressive phenotype