Abstract
To investigate the biological functions of TOP2A in HCC using bioinformatics and single-cell RNA sequencing (scRNA-seq) methods, and to explore the functional consequences of TOP2A in HepG2 and Huh-7 cells with TOP2A knockdown. This study aims to provide new theoretical insights and potential therapeutic targets for the early diagnosis and targeted treatment of HCC. Through the analysis of single-cell RNA sequencing data, the immunological significance of TOP2A and its relationship with the diagnosis and prognosis of HCC were studied. The expression levels of TOP2A in HCC and normal liver epithelial cells were validated using quantitative PCR (QPCR). siRNA technology was employed to interfere with TOP2A expression in HepG2 and Huh7 cells, and a TOP2A knockdown cell line was established. The transfection efficiency of siRNA was measured using QPCR and Western Blotting. The impact of TOP2A knockdown on the proliferation of HCC cells was assessed using the CCK8 assay. The effect of TOP2A on the migration ability of HCC cells was observed through a wound-healing assay. The influence of TOP2A knockdown on the invasive ability of HCC cells was examined using the Transwell assay. The effect of TOP2A knockdown on the apoptosis of HCC cells was evaluated using flow cytometry. This study integrated single-cell RNA sequencing and functional analyses identified TOP2A as a critical oncogenic driver in HCC. TOP2A expression was significantly upregulated in HCC tissues and strongly correlated with poor patient prognosis (P < 0.05). In vitro experiments showed that the efficiency of TOP2A knockdown mediated by siRNA was over 70% (P < 0.05), and it significantly inhibited the proliferation, invasion and migration of HCC cells (P < 0.05), promoted cell apoptosis (P < 0.05). Mechanistically, scRNA-seq revealed that TOP2A may promotes Tmem-to-Tex differentiation through the SPP1-CD44 axis. These findings position TOP2A as both a prognostic biomarker and a potential therapeutic target for overcoming immune evasion in HCC. TOP2A is significantly upregulated in HCC, and its expression level can serve as an independent prognostic biomarker. Knockdown of TOP2A can significantly inhibit the malignant phenotype of HepG2 and Huh7 cells. Moreover, the analysis shows that TOP2A may promote immune escape by modulating the SPP1-CD44 axis and remodeling the immune microenvironment. These findings not only reveal the crucial role of TOP2A in the progression of HCC, but also indicate that it may become a potential target for immunotherapy in HCC, providing a new theoretical basis for improving the precise treatment strategies for HCC patients.