Patient tissues obtaining

A total of 56 HCC tissues were obtained from patients who received liver biopsy partial hepatectomy or liver transplantation at First Affiliated Hospital of Xi’an Jiaotong University, and pathological data were obtained from the pathology department. Follow-up data within 5 years were collected, and survival analysis was conducted through Kaplan–Meier analysis with a log-rank test. Ethical approval was obtained from the ethics committee of First Affiliated Hospital, and informed consent was obtained from each patient.

Data collection and analysis, establishment of subtype model and other bioinformatics methods.

Gene expression data, DNA methylation data, and copy number data were obtained from UCSC Xena public database in a standard format. After patient code and gene ID matching, 372 patients with mRNA expression data of 447 PcG-related genes, DNA methylation data of 1109 PcG-related genes, and copy number data of 429 PcG-related genes were incorporated into subtype analysis. CancerSubtypes (version 1.8.0) were used to establish multi-omics HCC subtypes. Three clustering analyses (consensus clustering, CC, similarity network fusion, SNF, and non-negative matrix factorization, NFM) were performed, and the optimal clustering number was chosen according to the average silhouette value. Gene set variation analysis (GSVA) and oncoplot analysis were performed by GSVA (version 1.42.0) and maftools (version 2.10.05) on R software (version 3.5.1).

Immunohistochemistry assay (IHC)

HCC tissues from patients and subcutaneous tumors from nude mice were fixed in 4% paraformaldehyde for at least 48 h followed by dehydration and embedding in paraffin. Tissues were cut into sections for immunohistochemistry analysis. After deparaffinization with xylene, sections were subjected to antigen repair twice for 15 min at 100 °C with an interval cooling at room temperature for 5 min in citrate buffer (pH = 6.0). Tissue sections were blocked by 10% goat serum (Sigma-Aldrich) for 30 min and incubated with appropriately diluted primary antibody overnight at 4 °C (Additional file 1: Table S1). After being washed with PBS 3 times, tissue sections were incubated with the avidin–biotin complex for 20 min and were developed with diaminobenzidine. Finally, the tissue sections were counterstained with hematoxylin and mounted for observation. The proportion of high-positive (HP), positive (P), low-positive (LP), and negative cells were measured by the IHC profiler in Image J software (version 1.8.0). IHC scores were calculated by “IHC score = 300 × HP proportion + 200 × P proportion + 100 × LP proportion”.

Cell culture and treatment

Human HCC cell lines (MHCC-97L and Huh7) were purchased from the Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in DMEM medium supplemented with 10% fetal bovine serum, 100U/mL penicillin, and 100 μg/mL streptomycin to prevent contamination. Cells were cultured at 37 °C in a humidified atmosphere with 5% CO2. Oxaliplatin (HY-17371, MedChemExpress, 5 μg/ml), GSK126 (HY-19817, MedChemExpress, 2 μM), and PRT4165 (HY-19817, MedChemExpress, 10 μM) were dissolved in PBS or DMEM and diluted to an appropriate concentration when used.

For glucose deprivation treatment, HCC cells were seeded into 96-well plates and were maintained in a standard medium. After cell adhesion, the standard medium was replaced by a glucose-free culture medium with the palmitic acid supplement or not (10 μM). After 24 h of culture, cell viability was measured by CCK-8 assay.

RNA isolation, primer design and quantitative reverse transcription–polymerase chain reaction (qRT-PCR)

Total RNA from cells and patient tissues was extracted using Trizol (Invitrogen). Reverse transcription was performed according to the PrimeScript RT Master Mix protocol (TaKaRa, Mountain View, USA). Primers were designed using Primer-BLAST (NCBI) and were synthesized by GenePharma (Shang Hai, China). The primer sequences are shown in Additional file 1: Table S1. TB Green Premix Ex Taq (TaKaRa) was used to perform qPCR. The β-actin (ACTB) gene was used as the internal control.

Western blot

Total protein was extracted from tissues and cells by radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime). After concentration determination by BCA Protein Assay Kit (Beyotime), the protein was mixed with loading buffer (Beyotime) and heated to 100 °C for 10 min for denaturation. Protein (5 μg for the internal control and 20 μg for targets) was loaded into polyacrylamide gels and separated by electrophoresis. Protein was then transferred to polyvinylidene difluoride membranes. Membranes were incubated overnight at 4 °C with primary antibodies diluted in 10% milk (Additional file 1: Table S1). After being washed three times with PBS with 1% Tween (PBST), membranes were incubated with secondary antibodies for 30 min at room temperature. The membranes were washed 3 times with PBST before chemiluminescence.

Drug sensitivity measurement

Drug sensitivity measurement was based on the cell viability of HCC cells treated with oxaliplatin with a concentration of 0, 0.1, 0.5, 1.0, 5.0, 10.0, and 20.0 μg/mL, which was measured by CCK-8 assay. Cells were seeded into the 96-well plate with a density of 5000/well one day before pre-treatment of GSK126 or/and PRT4165. After 24 h of pre-treatment, HCC cells were washed with PBS 3 times. A total of 10 μL CCK-8 and 90 μL serum-free DMEM were added to each well, and the microplate was incubated at 37 °C for 1 h. The optical density at 450 nm was read with a 96-well multi-scanner plate reader (Biotech Instruments). Cell viability was calculated by the ratio of the optical density subtraction of treated and blank wells to the optical density subtraction of untreated and blank wells.

Gene overexpression and interference

Knockdown or overexpression of CBX2 in Huh7 and MHCC-97L cell lines was achieved by siRNA targeting CBX2 mRNA or overexpression plasmid. SiRNA and plasmid were designed and synthesized by GenePharma (Shanghai, China) according to a previous study and were delivered into cells by Lipofectamine 2000 (Additional file 1: Table S1) [23].

Immunofluorescence assay and lipid visualization

For lipid visualization, HCC cells were seeded in confocal dishes, after stable adherence cells were treated by GSK126 or PRT4165 for 24 h. Then, cells were stained by BODIPY (HY-125746, MedChemExpress) with concentration of 1 μM at 37 °C for 20 min and Hoechst 33,342 (Beyotime) at 37 °C for 5 min before observation by microscope. Particle analysis was performed by Images J software (version 1.8.0).

Cell apoptosis measurement

Cell apoptosis was measured by flow cytometry analysis. HCC cells were seeded into 6-well plates and pretreated with GSK126, PRT4165, or together before being treated with 5 μg/mL oxaliplatin. After 24 h of oxaliplatin treatment, HCC cells were harvested with PBS three times. Then, HCC cells were stained with Annexin V-PE and 7-aminoactinomycin D (7AAD, BD) according to the manufacturer protocol and analyzed for fluorescenceusing a flow cytometer (FACSCalibur, BD). The proportion of Annexin V-PE-positive cells was used to indicate the cell apoptosis rate.

Fatty acid oxidation measurement

Fatty acid oxidation measurement assay was based on the Fatty Acid Oxidation Assay (ab217602, Abcam, USA) and Extracellular Oxygen Consumption Assay (ab197243, Abcam, USA) according to manufacture’s protocol. HCC cells were seeded into 96-well plates and were maintained in standard medium overnight. After adherence, cells were pre-treated by GSK126 or PRT4165 and were maintained in glucose-deprivation (glucose-free) medium for 24 h. Then, cells were gently washed with fatty acid-free medium. Pre-warmed fatty acid measurement medium and extracellular O2 consumption reagent were added and wells were sealed by oil. The optical signal was measure after 60 and 120 min by multi-scanner plate reader. Fatty acid oxidation effect was calculated by signal ratio of negative control group with inhibitor-treated group subtracted by background of blank well.

In vivo studies

Athymic nude mice were purchased from the Laboratory of Animal Research Center of Xi’an Jiaotong University. The xenograft tumor model was established in mice by subcutaneous inoculation with 1 × 106 Huh7 cells. The oxaliplatin administration was set on the 20th day after inoculation of HCC cells (5 mg/kg). On the 30th day, the mice were killed and the tumors were harvested, weighed, and photographed. The protocol of animal experiments was approved by the Institutional of Animal Care and Use Committee at Xi’an Jiaotong University. The tumor volume was calculated by the formula: V = Length × Width2 × 0.5.

Statistical analysis

All analyses were performed based on GraphPad Prism (Version 8.0). Data represent mean ± SD from 3 repeated experiments. The Student’s t-test was used for comparing quantitative data. One-way ANOVA was used for testing differences across quantitative data of multiple groups. The ranked data were analyzed with the Chi-square test. Pearson correlation analyses were performed to analyze the correlation of gene expression.