Clinical specimens and cell lines

The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC), Gene Expression Omnibus and International Cancer Genome Consortium datasets were acquired from the SangerBox platform (http://vip.sangerbox.com/). Tissue microarray chips (164 tissue dots, including 78 tumour dots for further analysis) were obtained from Shanghai Outdo Biotech Company (Shanghai, China). The ethics committee of the Second Affiliated Hospital of Harbin Medical University approved this study protocol. HCCLM3 (CTCC-400-0193) and Hep3B (CTCC-001-0021) cell lines were purchased from MeisenCTCC. The cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% foetal bovine serum (FBS) and 1% antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin). All cell lines were cultured at 37 °C in a 5% CO2 incubator.

Lentivirus, stable cell line construction and quantitative real-time PCR

The lentiviral vector system and empty vector were purchased from GeneChem Corporation (Shanghai, China). Stable cell lines expressing the target gene or negative control were selected by adding 0.5 μg/mL puromycin into the medium. Total RNA was extracted and quantified according to the protocol of the RNA purification kit (Thermo Fisher Scientific, Waltham, MA, USA). Quantitative real-time PCR was performed using SYBR Green Power Master Mix (Promega, Madison, WI, USA). The following primers were used for quantitative reverse-transcription PCR: UCK2 forward: 5′-GCCCTTCCTTATAGGCGTCAG-3′; UCK2 reverse: 5′-CTTCTGGCGATAGTCCACCTC-3′; GAPDH forward: 5′-AGAAGGCTGGGGCTCATTTG-3′, GAPDH reverse: 5′-AGGGGCCATCCACAGTCTTC-3′.

HCC stem cell detection, cell proliferation and cell cycle assay

To evaluate HCC stem cell characteristics, 1000 cells were seeded into 24-well plates pre-treated with the CSwell 600 kit (Suzhou Jiyan Biotech Co., Ltd., Jinang City, China). After 24 h, the number of cell spheres was counted under a microscope. To examine cell proliferation, the cells were seeded into 96-well plates at 2 × 103 cells per well, and cell viability was measured using Cell Counting Kit-8 assays (Dojindo Molecular Technologies, Kumamoto, Japan) at different timepoints. For cell cycle tests, 4 × 104 cells were stained according to the protocol of the Cycle TESTTM PLUS DNA Reagent Kit (BD Biosciences, Franklin Lakes, NJ, USA) and analysed using flow cytometry (FC500, Beckman Coulter, Brea, CA, USA).

Transwell assay

The migration and invasion of HCC cells were evaluated in Transwell assays. Chambers with or without pre-coated Matrigel were placed in a 24-well plate. DMEM supplemented with 10% FBS was added to the lower chamber. Cells (5 × 104) suspended in 100 µL of DMEM without FBS were seeded into the upper chamber and cultured for 24 h.

Western blotting and co-immunoprecipitation (co-IP)

Cells were lysed in radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors. The proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After blocking with 5% skimmed milk at room temperature for 1 h, the membranes were incubated with primary antibodies overnight. The primary and secondary antibodies are listed in Additional file 5: Table S1. The membranes were then incubated with secondary antibodies for 1 h at room temperature, and the bands were detected using an enhanced chemiluminescence kit. For co-immunoprecipitation, the cells were incubated with pre-cold immunoprecipitation (IP) lysis buffer for 5 min and then transferred into a 1.5 mL tube for centrifugation at 13,000g for 10 min. The supernatant was collected, and the IP process was performed according to the protocol of the A Dynabeads protein G IP Kit (Thermo Fisher Scientific). Western blotting was performed as described above.

Immunohistochemical (IHC) staining

Deparaffinized tissue sections were stained with diaminobenzidine (DAB kit; Vector Laboratories, Burlingame, CA, USA) and counterstained with haematoxylin (Sigma, St. Louis, MO, USA) to visualize the immunoreaction product following the manufacturer’s protocol. The staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). The percentage scores were defined as 0, < 5%; 1, 5–25%; 2, 26–50%; 3, 51–75%; 4, > 75%. The histological score for each section was calculated using the following formula:

Immunofluorescence (IF) assays

The cells were seeded onto coverslips in six-well plates and incubated for 24 h. The cells were fixed with 4% paraformaldehyde and penetrated with 0.1% Triton-X-100. After incubation with the primary antibody for ICAM1 (ab222736, ABCAM), secondary antibody (Invitrogen, Carlsbad, CA, USA), and DAPI (Vector Laboratories) in sequence, images of the cells were acquired using a DMRA fluorescence microscope (20×, Olympus, Tokyo, Japan).

RNA-binding protein immunoprecipitation (RIP)

The cells were lysed using RIP assay buffer. The cell lysates were incubated with beads that had been pre-coated overnight with an antibody for UCK2 (ab241281, ABCAM). RNA was extracted from the samples according to the protocol of the RIP kit (Millipore, Billerica, MA, USA). Quality control and RIP sequencing were performed by GeneChem Corporation.

Determination of immune cell infiltration level and immune-related functional scores

Information on pan-cancer immune cell infiltration was obtained from TISIDB, an integrated repository portal for tumour–immune system interactions [19]. Using the CIBFERSORT database, we identified 22 immune cell types. The immune score, stromal score and tumour purity were generated using the R package “ESTIMATE”. Tumour immune dysfunction and exclusion (TIDE) and immunophenoscore analyses were performed as previously described [20, 21].

Statistical analysis

The correlation of gene expression and the survival of patients with HCC was determined using Lasso–Cox analysis with R package “glmnet”. Student’s t-test was used for comparisons between the two groups. Pearson’s correlation analysis was used to determine the linear relationship between the two groups. Multivariate analysis was performed using the Cox regression model. Kaplan–Meier curves were used to compare survival, and the log-rank test was used to compare survival between different groups. The results were considered as significant when p < 0.05.