目的

探讨金刚藤的主要成分菝葜皂苷元 （SAR）对肝癌的抗肿瘤作用及其潜在分子机制。

方法

采用SAR处理人肝癌细胞HepG2；运用CCK-8和EdU染色方法定量评估细胞的增殖活性；流式细胞术分析细胞的凋亡状况；Transwell实验检测细胞的迁移与侵袭能力；通过RNA测序 （RNA-seq）、GO、KEGG和PPI分析差异性表达基因功能、信号通路和蛋白相互作用。两组间比较采用独立样本t检验，多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。

结果

CCK-8实验显示，HepG2细胞的抑制率随SAR浓度的升高而升高，IC50值为22.5 μg/mL。与对照 （DMSO）比较，SAR处理抑制HepG2细胞的增殖，促进HepG2细胞凋亡，抑制细胞迁移[（40.00 ± 2.00）%比 （80.00 ± 5.00）%］和侵袭[（35.00 ± 2.00）%比 （70.00 ±4.00）%］（P 均 ＜ 0.01）。RNA-seq结果表明，SAR处理后，HepG2细胞基因表达发生变化：其中1 308个基因表达上调，2 966个基因表达下调；这些差异表达基因主要富集于细胞增殖和信号受体结合等生物学过程；KEGG分析发现差异表达基因主要富集于类固醇生物合成，补体及凝血级联反应通路，TNF和NF-κB等信号通路。PPI分析显示，CDK3、EPHA7、LRRK2、PRKCB、ANK3、TEK和ZNP70是SAR调控的靶基因。

结论

SAR可降低HepG2细胞的增殖、迁移和侵袭能力，促进其凋亡，其机制可能与TNF信号通路和NF-κB等信号通路相关。

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.