β‐Sitosterol is a natural compound with demonstrated anti‐cancer properties against various cancers. However, its effects on hepatocellular carcinoma (HCC) and the underlying mechanisms are not well understood. This study aims to investigate the impact of β‐sitosterol on HCC. In this study, we investigated the effects of β‐sitosterol on HCC tumour growth and metastasis using a xenograft mouse model and a range of molecular analyses, including bioinformatics, real‐time PCR, western blotting, lentivirus transfection, CCK8, scratch and transwell assays. The results found that β‐sitosterol significantly inhibits HepG2 cell proliferation, migration and invasion both in vitro and in vivo. Bioinformatics analysis identifies forkhead box M1 (FOXM1) as a potential target for β‐sitosterol in HCC treatment. FOXM1 is upregulated in HCC tissues and cell lines, correlating with poor prognosis in patients. β‐Sitosterol downregulates FOXM1 expression in vitro and in vivo. FOXM1 overexpression mitigates β‐sitosterol's inhibitory effects on HepG2 cells. Additionally, β‐sitosterol suppresses epithelial–mesenchymal transition (EMT) in HepG2 cells, while FOXM1 overexpression promotes EMT. Mechanistically, β‐sitosterol inhibits Wnt/β‐catenin signalling by downregulating FOXM1, regulating target gene transcription related to HepG2 cell proliferation and metastasis. β‐Sitosterol shows promising potential as a therapeutic candidate for inhibiting HCC growth and metastasis through FOXM1 downregulation and Wnt/β‐catenin signalling inhibition.

β-galactosidase, encoded by the lacZ gene in E. coli, can cleave lactose and structurally related compounds to galactose and glucose or structurally related products. Its activity can be measured using an artificial substrate, o-nitrophenyl-β-D-galactopyranoside (ONPG). Miller firstly described the standard quantitative assay of β-galactosidase activity in the cells of bacterial cultures by disrupting the cell membrane with the permeabilization solution instead of preparing cell extracts. Therefore, β-galactosidase became one of the most widely used reporters of gene expression in molecular biology to reflect intracellular gene expression difference. But the Miller assay procedure could not monitor the β-galactosidase reaction in real time and its results were greatly influenced by some operations in the Miller procedure, such as permeabilization time, reaction time and concentration of the cell suspension. A scanning method based on the Miller method to determine the intracellular β-galactosidase activity in E. coli Tuner (DE3) expressing β-galactosidase in real time was developed and the permeabilization time of cells was optimized for that. The comparison of 3 assays of β-galactosidase activity (Miller, colorimetric and scanning) was made. The results proved that scanning method for the determination of enzyme activity with using ONPG as substrate is simple, fast and reproducible.

INTRODUCTIONHepatocellular carcinoma (HCC), the primary form of hepatic carcinoma and a prevalent malignant tumor, is associated with a poor prognosis (Gentile et al., 2020). Various risk factors, such as cirrhosis, autoimmune hepatitis, HBV or HCV infection, nonalcoholic fatty liver disease, and alcohol abuse, contribute to HCC progression (Fujiwara et al., 2018; Kim and Han, 2012; Wang and Deng, 2023). The aggressive proliferation, metastatic capacity, and high drug resistance rates of HCC cells are major factors contributing to their invasive behavior (Llovet et al., 2021; Villanueva, 2019). Despite significant progress in clinical HCC treatment, the prognosis for patients remains grim due to rapid progression and early recurrence (Cai et al., 2022; Donadon et al., 2016; Wang and Wei, 2020). Thus, it is crucial to develop new drugs and identify novel therapeutic targets for HCC treatment.Forkhead box proteins constitute an evolutionarily conserved family of transcriptional regulators. Among them, forkhead box M1 (FOXM1) is known to be expressed in proliferating cells and associated with the activation of the mitotic program (Zuppo et al., 2023). FOXM1 plays a crucial role in cancer progression, influencing various steps such as Epithelial-Mesenchymal Transition (EMT), cell proliferation, migration, and premetastatic niche formation (Park et al., 2011; Raychaudhuri and Park, 2011; Zhang et al., 2017). In the context of HCC, FOXM1 has been found to be upregulated and contributes to the progression of HCC by directly regulating the expression of KIF4A (Hu et al., 2019). Notably, carfilzomib has demonstrated effectiveness in inhibiting FOXM1 expression in AFP-positive HCC cells, leading to the inhibition of tumor growth (Li et al., 2022). These findings underscore the significance of FOXM1 as a potential therapeutic target in HCC treatment.The canonical Wnt/β-catenin signaling pathway plays a pivotal role in regulating cell growth, differentiation, and migration (Gao et al., 2018; Liu et al., 2022). Central to this pathway is the β-catenin protein, which, upon translocation into the nucleus, interacts with the TCF/LEF-1 transcription factor complex to activate the expression of Wnt target genes, including C-myc, CyclinD1, MMP2, MMP7, and MMP9 (Yu et al., 2021). Dysregulation of this signaling cascade contributes to tumorigenesis and metastasis.β-Sitosterol, one of the most prevalent phytosterols, is widely distributed in vegetable oils, nuts, and traditional Chinese medicinal plants (Babu and Jayaraman, 2020). β-Sitosterol, one of the most prevalent phytosterols, is widely distributed in vegetable oils, nuts, and traditional Chinese medicinal plants (Khan et al., 2022). Numerous studies suggest that β-sitosterol hinders the malignant behavior of cancer cells by inhibiting their proliferation and migration, inducing apoptosis, and interfering with cellular metabolism (Awad et al., 2008; Bae et al., 2021). Despite the significant role of β-sitosterol in various diseases, its impact on HCC and the underlying mechanism remains unexplored. Thus, the objective of this study was to investigate the inhibitory effect of β-sitosterol on HCC progression by regulating the FOXM1-mediated Wnt/β-catenin signaling pathway.