Exploration of the Key Mechanism by Which Kaempferol Inhibits Glycolysis in Hepatocellular Carcinoma Cells via the AKT – mTOR Pathway

ABSTRACT Hepatocellular carcinoma (HCC) remains a lethal malignancy with limited therapeutic options. Kaempferol shows potential in suppressing HCC progression via glycolysis regulation, yet its molecular targets and mechanisms are unclear. Transcriptomic data were analyzed to identify HCC‐related DEGs. Intersection with kaempferol's glycolysis‐related targets yielded candidate genes. A prognostic risk model was constructed and validated by ROC curves and survival analysis. GSEA and single‐cell RNA sequencing explored mechanisms and cellular heterogeneity. Mendelian randomization assessed causality between prognostic genes and HCC. After HCC cells were treated with different concentrations of kaempferol, the effects of kaempferol on proliferation, invasion, and migration of HCC cells were detected by CCK‐8, cell clone formation, and Transwell cell scratch healing experiments. The expression levels of key proteins in the AKT–mTOR signaling pathway and glycolytic rate‐limiting enzymes were detected by Western blot. The spectrophotometric method was used to detect the effect of kaempferol on glucose uptake and lactate production of HCC cells. Finally, the impact of knocking down CA9 on glucose uptake and lactic acid production in liver cancer cells was analyzed. The prognostic risk model identified GRK6 , ABCC1 , CA9, and CDK5R1 as prognostic genes. GSEA implicated cell cycle and PI3K‐Akt pathways in HCC progression driven by these genes. Single‐cell analysis revealed pronounced upregulation of prognostic genes in hepatocytes. Finally, MR analysis confirmed that CDK5R1 was a risk factor for the incidence of HCC. Kaempferol significantly inhibited the proliferation, invasion, and migration of MHCC97H and Huh7 cells, as well as the expression levels of PKM2 , HK2 , p‐AKT , p‐mTOR , and p‐RPS6 . Kaempferol significantly reduced glucose uptake and lactic acid production in MHCC97H and Huh7 cells. Knockdown of CA9 inhibited the uptake of glucose and lactic acid production, along with suppressing the expression of HK2 and PKM2 in MHCC97H and Huh7 cells. This study found that kaempferol targeted the AKT–mTOR pathway and downregulated the CA9 gene, thereby modulating glycolysis and ultimately suppressing the proliferation, invasion, and migration of hepatocellular carcinoma.