Abstract:

Objective

To investigate the anti-tumor effects of sarsasapogenin （SAR），a major constituent of Smilax china L.， on hepatocellular carcinoma and its potential molecular mechanism.

Methods

In this study， HepG2 cells were treated with SAR. Cell proliferation activity was assessed using the CCK-8 assay and EdU staining. Cell apoptosis was determined by flow cytometry. Cell migration and invasion were detected by transwell assay. Dfferential expressed gene functions， signaling pathways， and protein interactions were analyzed through RNA sequencing， GO，KEGG， and PPI analysis. The independent sample t test was used for comparison between two groups，the one-way analysis of variance was used for comparison between multiple groups， and the LSD-t test was used for pairwise comparison between mutiple groups.

Results

The CCK- 8 experiment revealed that SAR treatment significantly inhibited HepG2 cell proliferation with an increase of the concentration， and the IC50 value was 22.5 μg/mL. Compared to control （DMSO）， SAR inhibited cell proliferation， increased cell apoptosis level significantly， and led to a significant decrease in cell migration [（40.00 ± 2.00）% vs （80.00 ± 5.00）%］and invasion rates [（35.00 ± 2.00）% vs（70.00 ±4.00）%］ （all P ＜ 0.01）. RNA-seq showed significant changes in gene expression in HepG2 cells after SAR treatment. 1 308 genes were upregulated， while 2 966 genes were downregulated.The differentially expressed genes were significantly enriched in biological processes such as cell proliferation and signal receptor binding. Further KEGG analysis revealed that the differentially expressed genes mainly enriched in steroid biosynthesis， complement and coagulation cascade，TNF， MAPK， and NF-κB pathway. The hub genes CDK3， EPHA7， LRRK2， PRKCB， ANK3， TEK and ZNP70 were identified as the targets of SAR by PPI network analysis.

Conclusion

SAR could reduce the proliferation， migration and invasion and promote apoptosis of HepG2 cells. The underlying mechanism may involve the TNF signaling pathway， the NF-κB signaling pathway， and other related signaling pathways.

Key words: Sarsasapogenin, HepG2 cells, Anti-tumor, Signaling pathway