HCC and adjacent normal tissues (distance, >5cm) were collected from five patients (age range from 51 to 73, five patients were all males) with HCC from September to November 2024 in The First People's Hospital of Anqing (Anqing, China) during routine surgery. The present study was approved by the Research Ethics Committee of The First People's Hospital of Anqing (approval no. 20240077), and written informed consent was obtained from all participants prior to participation.

Gene expression profiles for datasets GSE84402, GSE26538 and GSE141090 were retrieved from the Gene Expression Omnibus (GEO, ncbi.nlm.nih.gov.cn). The Xiantao Academic website (20) was used for generating Venn diagrams and analyzing the association between immune infiltration and CYP8B1 expression. The association between CYP8B1 expression and immune cell infiltration was analyzed based on ssGSEA algorithm provided in R package (version 4.2.1; cran.r-project.org/). The expression of CYP8B1 and the content of immune cells was obtained from The Cancer Genome Atlas (TCGA) database [TCGA-liver hepatocellular carcinoma (LIHC, the URL was: portal.gdc.cancer.gov). The association between the expression of CYP8B1 and the content of immune cells was analyzed by Spearman's test and to further show the relationship between CYP8B1 and immune infiltration. The content of the immune cells can indicate the degree of immune infiltration. The survival and expression analyses of CYP8B1 were performed using CYP8B1 expression data and clinical information from the UALCAN (ualcan.path.uab.edu) (21) and Gene Expression Profiling Interactive Analysis (GEPIA) platform (gepia.cancer-pku.cn) (22). Cox regression analysis and Kaplan-Meier plots for TCGA datasets was performed using RStudio software (version 4.2.1) to investigate the association between CYP8B1 expression and cancer prognosis, including overall survival in LIHC subclinical groups. Additionally, the UALCAN and GEPIA database were also used to determine the association between CYP8B1 expression and overall survival. The heatmap of positively and negatively associated genes with CYP8B1 in LIHC was analyzed by UALCAN database to show the top 24 associated genes. Furthermore, gene analysis of positively and negatively associated genes with CYP8B1 in LIHC were performed using the Metascape website (metascape.org) to show the pathway and biological progress of these genes (23). Gene expression of CYP8B1 in HCC cell lines was performed using the Depmap website (depmap.org/portal/).

HCC cell lines [Huh7 (cat. no. SCSP-526) and Hep3b (cat. no. SCSP-5045)] were obtained from Shanghai Institutes of the Chinese Academy of Sciences. The cells were maintained in high-glucose DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin (complete medium) at 37°C in a humidified incubator with 5% CO₂.

The recombinant CYP8B1 plasmid was constructed by cloning into the pcDNA3.0 (Shanghai GeneChem Co., Ltd.) vector and transfected into HCC cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), following the manufacturer's protocols. A total of 2 µg/well overexpressed CYP8B1 plasmid (oe-CYP8B1) and empty vector (negative control, NC) was transfected into HCC cells in 6-plate well for 6 h for duration in at 37°C, the medium was replaced with complete medium. After 48 h, the cells were collected for subsequent experimentation.

The CCK-8 assay were performed to assess cell proliferation as previously described (20). Briefly, transfected 5000 HCC cells were seeded into every well of 96-well plates. Cell proliferation was measured using CCK-8 (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's instructions after incubation for 1, 2, 3 and 4 days. All the experiments were performed in triplicate.

EdU incorporation assay was used to measure cell proliferation rate as previously described (20). The proliferation rate was calculated following the manufacturer's instructions using the BeyoClick™ EdU-555 EdU kit (Beyotime Institute of Biotechnology). Each EdU incorporation experiment was performed ≥3 times.

Cell apoptosis was assessed using the TUNEL assay kit (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's instructions, as previously described (24). The experiments were repeated ≥3 times.

Protein samples from patient tissue and tumor cells) were lysed in RIPA buffer supplemented with fresh protease and phosphatase inhibitor cocktails (Beyotime Biotech Co., Ltd.). The protein concentration was determined using the BCA assay (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of proteins (20 µg/lane) were separated by10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 3% BSA (Beyotime Biotech Co., Ltd.) in 10 mM Tris-HCl (pH 7.4) containing 0.05% Tween-20 to prevent non-specific binding. Membranes were incubated with primary antibody at 4°C for 12 h, followed by incubation with a corresponding peroxidase-conjugated secondary antibody (Abcam; Table SI) at room temperature for 2 h. Immunoreactive bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce; Thermo Fisher Scientific, Inc.) and band density was quantified using a Versadoc Imaging System Model 3000. Each experiment was conducted ≥3 times.

Total RNA was extracted from HCC cell lines using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. RT-qPCR was performed as previously described (25). Briefly, 1 µg total RNA was reverse-transcribed using random primers and Primescript reverse transcriptase (Vazyme Biotech Co., Ltd.). RT protocol was as follows: 37°C for 15 min, 85°C for 5 sec, 4°C forever. qPCR for the target genes was conducted using the SYBR-Green qPCR kit (Vazyme Biotech Co., Ltd.) on a fluorescent temperature cycler (ABI 7500 Real-Time PCR system; Thermo Fisher Scientific, Inc.). The primers were as follows: CYP8B1: Forward, 5′-ACCTGAGCTTGTTCGGCTAC-3′ and reverse, 5′-CGGAGAGCATCTTGTGAAAG-3′ and β-actin: Forward, 5′-ATCGTGCGTGACATTAAGGAGAAC-3′ and reverse, 5′-AGGAAGGAAGGCTGGAAGAGTG-3′. The thermocycling conditions were as follows: Initial denaturation at 95°C for 5 min, followed by 45 cycles of amplification at 95°C for 15 sec and annealing at 60°C for 1 min. Each experiment was performed ≥3 three times. Relative levels of mRNA expression were obtained via the 2^−ΔΔCq method (26).

All statistical analyses were performed using SPSS 18.0 software (SPSS Inc.). Data are presented mean ± SD, and independent experiments repeats three times. Differences between categorical variables were analyzed using the independent sample unpaired or paired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

HCC gene expression profiles from GEO datasets, including data from human, mice and rat studies, were used to identify differentially expressed genes (DEGs) using a Venn diagram. The analysis revealed one up- (CDK1) and four downregulated DEGs [CYP8B1, MFSD2A (major facilitator super family domain containing 2a), VIPR1 (Vasoactive intestinal peptide receptor 1), THRSP (Thyroid hormone-responsive protein)] in HCC compared with normal tissues (Fig. 1A-C). Except for CYP8B1, these DEGs have been studied in HCC (27–29); therefore, the present study investigated CYP8B1. In TCGA database, CYP8B1 expression was significantly lower in HCC tumor tissues compared with normal tissues, as shown on the UALCAN (Fig. 1D) and the GEPIA platform (Fig. 1E). Consistently, GTEx and TCGA databases demonstrated reduced CYP8B1 expression in HCC compared with normal tissue (Fig. 1F). In the GSE84402 dataset, CYP8B1 levels were significantly lower in HCC tissues than in corresponding normal tissue (Fig. 1G). Additionally, tumor and adjacent normal tissue samples were collected from five patients with HCC. Immunoblotting analysis showed that CYP8B1 expression was significantly lower in tumor tissues compared with adjacent normal tissues (Fig. 1H and I).

To evaluate the prognostic significance of CYP8B1 in HCC, UALCAN and GEPIA databases were used. Patients with HCC with low CYP8B1 expression exhibited worse overall survival outcomes (Fig. 2A and B). Decreased CYP8B1 expression was associated with poorer OS in specific clinical subgroups, including those with AFP (α-fetoprotein) levels >400 ng/ml, individuals aged ≥60 years, male patients, patients taller than 170 cm, Asian patients and patients with residual tumor (R0; Fig. 2C-H).

Given the key role of tumor-infiltrating lymphocytes in cancer progression and their impact on patient prognosis (30), along with the potential oncogenic role of CYP8B1 in HCC, the relationship between CYP8B1 expression and the degree of immune infiltration in HCC was investigated. We detected the association between CYP8B1 expression and different immune cells content in TCGA database and found that CYP8B1 expression was positively associated with content of the following immune cells such as T helper cell (Th17) cells, neutrophils, central memory T cell) cells, Tregs (Regulatory T cells), DC (Dendritic cells), Tgd (T γΔ) cells and eosinophils. Conversely, CYP8B1 expression showed a significant negative correlation with content of the following immune cells such as Th1 cells, active Dendritic cells), B cells (Bone marrow-dependent lymphocyte), interdigitating Dendritic cells), Th (T helper cells) and T (Thymus-dependent lymphocyte) cells, macrophages and effective memory T cell), Th2 (T helper cell 2), TFH (Follicular helper T cell) and NK (Nature killer) CD56 bright cells. (Fig. 3A-G). Furthermore, we selected 17 types immune cells which was significantly associated with expression of CYP8B1 and found that the low and high expression of CYP8B1 has significant differences in all the 16 immune cells except for eosinophils (Fig. 3H).

To investigate the role of CYP8B1 in HCC, its expression levels were searched in public datasets (depmap.org/portal/). CYP8B1 expression was high in HepG2 and low in Huh7 and Hep3B cells (Table SII). To study the functional effects, CYP8B1 was overexpressed in Hep3B and Huh7 cells by transfection with a CYP8B1-overexpression plasmid. Following transfection, both mRNA (Fig. 4A and C) and protein levels (Fig. 4B and D) of CYP8B1 were significantly increased in the two HCC cell lines.

To evaluate the effect of CYP8B1 on cell proliferation, CCK-8 (Fig. 5A) and EdU assays (Fig. 5B and C) were performed in cells overexpressing CYP8B1. Both assays showed that CYP8B1 overexpression significantly decreased the proliferation of Huh7 and Hep3b cells (Fig. 5D-F).

To investigate the effect of CYP8B1 on apoptosis, TUNEL assay was performed. CYP8B1 overexpression markedly increased the apoptosis rate in Huh7 (Fig. 6A and B) and Hep3b cells (Fig. 6C and D).

To explore the mechanisms of CYP8B1 in HCC, the top 24 genes positively and negatively correlated with CYP8B1 expression in LIHC were identified using the UALCAN platform (Fig. 7A and B) and submitted to the Metascape platform for pathway enrichment analysis (Fig. 7C and D). This revealed two key pathways potentially regulated by CYP8B1: ‘Regulation of PLK activity at G2/M transition’ and ‘intrinsic pathway for apoptosis’ (Fig. 7C). As CYP8B1 overexpression inhibited cell proliferation and promoted apoptosis, the present study identified genes from PubMed involved in the G2/M transition and apoptosis. YWHAZ (Tyrosine 3/tryptophan 5 monooxygenase activation protein ζ) downregulation induces G2/M transition and apoptosis (23,24). Expression of YWHAZ was negatively associated with CYP8B1 in LIHC (Fig. 7A). Immunoblot assay was used to clarify the expression of YWHAZ after CYP8B1 overexpression, as well as the expression of downstream factors of YWHAZ, including Cyclin B1, CDK1, Bax and Bcl2 (Fig. 7E and F) in Huh7 and Hep3b cells. YWHAZ was significantly downregulated following CYP8B1 overexpression. Downstream factors of YWHAZ, including Cyclin B1 and CDK1 which was involved in the G2/M cell cycle was significantly downregulated. Apoptosis-related genes Bax was upregulated and Bcl2 was downregulated after CYP8B1 overexpression. The results suggested that CYP8B1 mediates G2/M arrest via the YWHAZ/CyclinB1/CDK1 axis and apoptosis primarily via the YWHAZ/Bax/Bcl2 axis.