Database analysis
TCGA-LIHC gene expression data were downloaded from the TCGA data portal (https://tcga-data.nci.nih.gov/tcga/ ) and three gene expression profiles (GSE102079 , GSE121248 and GSE14520) as raw data. The Human Protein Atlas web (http://www.proteinatlas.org/) was used to obtain relevant immunohistochemical staining images of COPB2 and YAP1 in HCC tissues.
WGCNA analysis
Weighted gene co-expression network analysis (WGCNA) of GSE02079 and GSE121248 expression matrices using the R package "WGCNA".
Patient and clinical samples
We retrospectively analyzed two independent cohorts comprising HCC 214 patients. In the training group, 114 HCC patients from the Affiliated Hospital of Nantong University from 2012-2017 were randomly selected. Moreover, 100 HCC patients from the Affiliated Tumor Hospital of Nantong University from 2011-2016 were randomly selected as the validation group. Cases are selected according to the following criteria: no previous anti-cancer related treatment prior to surgery; pathological diagnosis of HCC; availability of resected tissue and follow-up data. Patients receive a physical examination, laboratory diagnosis, and imaging twice a year after surgery. Most patients undergo TACE after relapse. Overall survival (OS) was measured from the date of surgery to the date of death or last follow-up. For each patient, the following clinicopathological information was collected: age, gender, hepatitis B virus (HBV), presence of cirrhosis, serum alpha-fetoprotein (AFP) level, tumor number, tumor size, degree of tumor differentiation, vascular tumor thrombus and lymph node metastasis. The staging of tumors is defined according to the American Joint Committee on Cancer/International Union Against Cancer's Tumor Lymph Node Metastasis (TNM) classification system. Written informed consent was obtained from all participating patients for the use of this clinical information, and the project was approved by the ethics committees of both hospitals.
Immunohistochemistry
The tissue microarrays (TMA) sections are dewaxed in xylene and rehydrated through a graded alcohol series. Pressure cook the sections in alkaline buffer (pH=9.0) for 3 minutes. After cooling at room temperature, immerse in 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity. The sections were then incubated with rabbit polyclonal anti-COPB2 antibody (1:1000, Abcam, Cambridge, UK), YAP1 monoclonal antibody (1:200, Abcam, Cambridge, UK) overnight at 4℃. After washing three times with PBS, the sections were incubated with secondary antibodies (1:4000, Abcam, Cambridge, UK) for 20 min at room temperature, and then diaminobenzidine (DAB) solution was applied. Finally, sections were re-stained with hematoxylin, dehydrated and fixed. All microarrays were scored independently by two experienced pathologists. COPB2 scoring criteria: The percentage of positive cells was evaluated on a scale of 0–3:0(0–25%),1 (26–50%), 2 (51–75%), or 3(76–100%). Classified as low expression group:0-1 point or high expression group; 2-3 points. YAP1 scoring criteria, with a percentage of positive nuclei greater than 10% recorded as high expression and less than 10% recorded as low expression.
Western blot
HCC cells were lysed in cell lysis buffer on ice (Beyotime, Shanghai, China). The protein concentration was determined using the bicinchoninic acid assay (Beyotime, Shanghai, China). Electrophoresis was then performed on SDS-PAGE gels and transferred to PVDF membranes. The following antibodies were used in this study: COPB2 (1:1000, rabbit, Abcam, Cambridge, UK), YAP1 (1:500, rabbit, Abcam, Cambridge, UK), pLATS1(1:500, rabbit, Cell Signaling Technology, Massachusetts, USA), pYAP1 (1:500, rabbit, Cell Signaling Technology, Massachusetts, USA), GAPDH (1:5000, mouse, Abcam, Cambridge, UK).
Cell culture and vectors
The human HCC cell lines Huh7 and SK-hep1 were purchased from the Academy of Sciences Committee of the China Cell Resource Centre (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle 92 medium (DMEM, Hyclone, Logan, Utah, USA) containing 10% fetal bovine serum (FBS, Gibco, Grand 93 Island, NY, USA) at 37°C in a humidified atmosphere of 5% CO2. HCC cells in the exponential growth phase were used for subsequent experiments. The COPB2 siRNA was designed by Polyplus (Sense: CCCAUUAUGUUAUGCAGAUTT; Antisense‐1: AUCUGCAUAACAUAAUGGGTT). YAP1/pcDNA3.1 was constructed and the sequence was examined by GENECHEM. For transient expression, plasmids were transfected with Lipofectamine 2000 (Invitrogen, Shanghai, China) for 24 hours, after which the cells were processed with the indicated reagents as described above.
Cell Counting Kit-8 (CCK-8) assay
The Huh7 and SK-hep1 cells were digested with 0.25% trypsin and diluted to a 5×104 cells/ml concentration. The cells were counted using a cell counter(Thermo Fisher Scientific, Massachusetts, USA) and the cell suspension was inoculated into 96-well plates at a density of 2000 cells/well. Add 10μL CCK-8 solution (NCM, Suzhou, China) to each well, and culture at 37℃ for 2 hours. Cell viability was determined by measuring the absorbance at 450nm.
Clone formation assay
Briefly, Huh7 and SK-hep1 cells (2000 cells/well) were inoculated in 6-well plates and incubated at 37℃. The medium was changed every three days and observed cell growth. A total of 14 days after inoculation of cells, colonies with a cell count of > 50 were observed under a microscope. Cells were washed with PBS and fixed in 4% paraformaldehyde (Biosharp, Hefei, China) for 20 minutes at room temperature. Fixed cells were stained with 1 ml of Crystal Violet Staining Solution (Beyotime, Shanghai, China) for 10 minutes, washed with PBS and air dried at room temperature. The cells were photographed and the number of clones visible was counted.
Transwell assay
Migration assay: Transwell chambers were placed in 24-well plates. 2 × 105 Huh7 and SK-hep1 cells starved in 200 μL of serum-free medium were added to the upper chamber, and 500 μL of medium with 10% serum was added to the lower chamber. After 24 hours of incubation at 37℃ and 5% CO2, the Transwell chambers were first fixed in paraformaldehyde for 30 minutes and then stained with Crystal Violet Staining Solution for 10 minutes. The cells were photographed and counted under the microscope, with three replicates per group.
Invasion assay: Melt Matrigel at 4℃ overnight and dilute with pre-cooled serum-free DMEM medium. Add 100µL of diluted Matrigel to the upper chamber. The remaining steps are the same as those in the Transwell migration assay
Immunofluorescence
Huh7 and SK-hep1 cells were inoculated in 24-well plates for 24 hours. Cells were then fixed in 4% paraformaldehyde for 15 minutes and permeabilized for 10 minutes. After being closed with 5% BSA for 1 hour, the cells were incubated overnight at 4°C with YAP1 antibody (1:100, Abcam, Cambridge, UK) and then Alexa Fluor 594 anti-rabbit IgG secondary antibody (Invitrogen, Shanghai, China). Furthermore, cell nuclei were stained with DAPI (Thermo Fisher). The images were taken under a fluorescent microscope.
Statistical analysis
Differences in expression were analyzed by paired t-test. Clinicopathological characteristics were assessed using the chi-square test. P < 0.05 was considered a statistically significant difference. Statistical analyses were performed using the SPSS 26.0 statistical package (SPSS, Inc, Chicago, IL) and GraphPad Prism version 7.01 (GraphPad Software, Inc, La Jolla, CA). Nomogram was developed using the Regression Modelling Strategies package in R (version 3.0.2; R Package "rms" for nomogram establishment; www.r-project.org). ANN was built using SPSS Clementine version 12.0 software for Windows (IBM Corporation).