Data acquisition and bioinformatic analysis
The RNA sequencing data of the HCC cohort, including 371 HCC and 50 adjacent non-tumorous samples, was downloaded from The Cancer Genome Atlas Program (TCGA) database (https://portal.gdc.cancer.gov/). The HCC datasets, including GSE121248 (70 HCC and 37 normal samples), GSE136247 (39 HCC and 30 normal samples), GSE41804 (20 HCC and 20 normal samples), and GSE14520 (247 HCC and 241 normal samples) were screened from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). Concerning the HCC cohort from TCGA database, the GEPIA tool (http://gepia.cancer-pku.cn/) was operated to analyze the correlation of VCP expression and survival probability following the criteria of the best cut-off algorithm based on VCP transcriptional level in patients. Meanwhile, the UALCAN tool (http://ualcan.path.uab.edu/) was performed to obtain all candidate genes co-expressed with VCP according to Pearson’s correlation coefficient.
Gene set enrichment analysis
Gene set enrichment analysis (GSEA) is a computational algorithm utilized to assess whether a previously defined gene set indicates a statistical significance and concurrent difference between two biological phenotypes . The Kyoto Encyclopedia of Genes and Genomes (KEGG) gene sets were retrieved from the Molecular Signatures Database (MSigDB, version 7.5.1). HCC patients from TCGA database were divided into the high and low expressed groups according to the median transcriptional value of VCP and HMGB1, respectively. The normalized enrichment score (NES) and false discovery rate (FDR) were determined by GSEA software (version 4.1.0). FDR value < 0.05 was thought of as statistical significance.
Function annotation and protein–protein interaction network
Gene ontology (GO) analysis, including biological process (BP), cell components (CC), and molecular function (MF), and pathway enrichment including KEGG and Reactome panels, were performed using the Metascape online tool (https://metascape.org/). The protein–protein interaction (PPI) network was constructed in the STRING database (https://string-db.org/) and visualized via Cytoscape software (version 3.8.2). The hub genes were identified using the Cytohubba plug-in.
Cell culture and transfection
Human HCC cell lines of Huh7 and MHCC-LM3 cells, as well as 293T cells, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The immortalized hepatocyte cell line L02 was obtained from the China Center for Type Culture Collection (CCTCC, China). Huh7, MHCC-LM3, and 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA), and L02 cells were maintained in RPMI medium (Gibco). All were supplemented with 10% fetal bovine serum (FBS, Gibco, USA), penicillin (100 U/ml), and streptomycin (100 μg/ml), and cultured in a humidified incubator with 5% CO2 at 37 °C.
The stable cell line was constructed with lentivirus transduction using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. Huh7 cells were transfected by PLKO.1-puro-VCPshRNA (VCP-sh1 and VCP-sh2) or PLKO.1-puro-nonspecific shRNA (as control), MHCC-LM3 cells were treated with PLV-EGFP-puro-Myc-VCP or PLV-EGFP-puro plasmid (as control). Then, HCC cells with successful lentivirus transfection were screened by puromycin for 10 to 14 days. Small interfering RNA (siRNA) and various plasmids were transfected into cells using Lipofectamine 2000. HMGB1-siRNA target sequences (5′ to 3′) #1: CCCGTTATGAAAGAGAAATTT. #2: GGAGGAAGATGAAGAAGATTT. VCP-siRNA target sequences (5′ to 3′) #1: GAATAGAGTTGTTCGGAAT. #2: GGCCAAAGCCATTGCTAAT.
Western blot and antibodies
Whole-cell lysates were collected using the protein lysis buffer containing proteinase and phosphatase inhibitors. The BCA assay was operated to measure the protein concentration. The protein was separated by 12% or 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels. The gel was then transferred to polyvinylidene difluoride (PVDF) membranes (0.25 μm, Millipore, Billerica, MA, USA), and blocked with 5% fat-free dry milk resolved in TBST buffer. The membranes were then washed three to five times (5 min/time) and incubated with the indicated primary antibodies including VCP (2648, CST, USA), HMGB1 (ab18256, Abcam, USA), E-cadherin (20874-1-AP, Proteintech, China), β-catenin (51067-2-AP, Proteintech, China), Snail (sc-271977, Santa Cruz, USA), Twist2 (66544-1-Ig, Proteintech, China), PI3K (4249, CST, USA), AKT (10176-2-AP, Proteintech, China), pAKT (66005-1-Ig, CST, USA), mTOR (20657-1-AP, Proteintech, China), pmTOR (5536, CST, USA), ub (sc-8017, Santa Cruz, USA), GAPDH (60004-1-Ig, Proteintech, China), Myc-tag (60003-2-Ig, Proteintech, China), His-tag (12698, CST, USA), and Flag-tag (66008-3-Ig, Proteintech, China). The bound antibodies were examined using enhanced chemiluminescent reagents (34577, Thermo Fisher, USA) after incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (FSM0075 and FSM0056, Fushen, China). The relative protein quantification was analyzed using ImageJ software.
Quantitative real-time PCR (qPCR)
Total RNA was extracted using TRIzol reagent (15596018, Invitrogen, USA) following the manufacturer’s instructions. Reverse transcription (PrimeScript RT reagent Kit, RR047A, Takara, Japan) and SYBR (1725124, Bio-Rad, USA) green-based real-time PCR were performed. GAPDH expression was regarded as endogenous control, and the value of 2−ΔΔCT was adopted to determine the relative gene transcriptional expression. The qPCR primers included VCP forward: 5′-CTGGAGCCGATTCAAAAGGTG-3′, reverse: 5′-ACACTGTGTCACCTCGGAAC-3′. HMGB1 forward: 5′-GCGAAGAAACTGGGAGAGATGTG-3′, and reverse: 5′-GCATCAGGCTTTCCTTTAGCTCG-3′.
Forty-four HCC samples and adjacent non-tumorous tissues from HCC patients who underwent surgical resection were collected at Xiangya Hospital, Central South University (CSU), Changsha, China, between March 2017 and March 2020. Written informed consent was obtained from all patients. This project was approved by the Medical Ethics Committee of the Xiangya Hospital, CSU. Immunohistochemistry was performed as described previously . The VCP expressed level was evaluated using Image J software, and semi-quantitatively scored as highly positive (4), moderately positive (3), low positive (2), and negative/undetectable (1) staining.
Mass spectrometry (MS)
The LC-ESL-LTQ-Orbitrap-MS method was used to identify VCP-interacting proteins as described in a previous study . Briefly, the protein bands were cut from the gel and transferred to 100 mM NH4HCO3 with 50% acetonitrile for excising and destaining. Subsequently, the proteins were reduced, alkylated, and dried in a vacuum centrifuge. The gel pieces harboring proteins were incubated in digestion solution at 37 °C for 18–24 h. The tryptic peptide mixture was purified with a ZipTipC18 microcolumn (ZTC18S096, Millipore, Germany) and subjected to separation on a Pep Map C18 trap column (75 μm, 15 cm) with column flow rates of 200 nL/min. MS/MS analysis of the seven strongest ions in the LTQ was conducted using MS and MS spectra. Next, Xcalibur and Proteome Discoverer software were used to analyze the MS data.
Cells were cultured in twelve-well plates with coverage of glass coverslips up to 30–40% confluency. The cells were washed and fixed with 4% paraformaldehyde before blocking with 5% BSA containing 0.1% Triton-100. Then, the cells were incubated with mouse HMGB1(SAB1403925, Sigma, USA) and rabbit VCP (2648, CST, USA) primary antibodies at 4 °C overnight. Next, the cells were stained with Alexa Fluor 488-conjugated anti-mouse IgG (A-21202, Thermo Fisher, USA) or Alexa Fluor 594-conjugated anti-rabbit IgG (A-11012, Thermo Fisher, USA) for 1 h at room temperature. After intermediate washes, fluorescent signals were detected under a confocal microscope (Olympus, Tokyo, Japan).
Cells were lysed in IP specific RIPA buffer (P0013D, Beyotime, China) and centrifuged for 20 min at 12,000g. The lysed samples were then incubated with protein A/G agarose beads (sc-2003, Santa Cruz, USA) for 2 h at 4 °C to prevent non-specific binding and spin. Subsequently, the proteins were incubated with specific antibodies or the same species of IgG (1 μg antibody: 1 mg cellular protein) overnight at 4 °C. Then, protein A/G agarose (20–80 μL) was added again for 3 h incubation, followed by extensive washing with RIPA buffer. Finally, the precipitated proteins were eluted by boiling in the 2 × SDS sample buffer and subjected to SDS-PAGE.
GST Sefinose Resin (C600031, Sangon Biotech, China) was used to purify the GST-tagged VCP protein complex. The purified proteins were examined using Coomassie blue staining. The lysate from MHCC-LM3 cells was incubated with GST-tagged proteins at 4 °C overnight with gentle rotation. The precipitated pellets were washed to elute resins and bound proteins were analyzed by SDS-PAGE.
Cell proliferation and apoptosis assays
For Cell Counting Kit-8 (C0038, Beyotime, China), cells were seeded at a density of 1000 cells/well into 96-well plates and cultured for 24 h, 10 μL CCK8 was added to each well and incubated for 2 h. The absorbance at 450 nm was measured using a PerkinElmer spectrophotometer. For the EdU Cell Proliferation Kit (C0078S, Beyotime, China), appropriate cells were seeded into 24-well plates and incubated for 24–36 h. EdU (20 μM) was added to each well and incubated for 2 h. Then, the cells were fixed with 4% paraformaldehyde and incubated with phosphate-buffered saline (PBS) containing 0.3% Triton X-100. Next, the cells were incubated with 0.5 mL Click Additive solution for 30 min followed by the nuclei stained with DAPI (1:1000) for 10 min in the dark. The fluorescence was examined under a fluorescence microscope.
For the apoptosis assay, the cells were digested with trypsin and washed twice with sterile PBS. Next, the cells were centrifuged for 5 min at 1000g. Then, cells were incubated with Annexin V labeled with PE and 7-Amino-Actinomycin (7AAD) for 15 min (559763, BD Biosciences, USA) at room temperature in the dark. Apoptotic cells were screened by flow cytometric analysis.
Wound-healing, migration, and invasion assay
For the wound-healing assay, 1 × 10 6 cells were seeded into six-well plates. When the cells reached 90% confluence, the cell layer was scratched with a 10 μL sterile plastic tip and cultured for 72 h in serum-free medium. Images were taken under a microscope at the indicated time point to evaluate the healing rate of gap closure.
For the transwell system, 5 × 104 cells were plated in the upper compartment of a transwell chamber (8 μm size, 3422, Corning, USA) in a serum-free medium. The upper chamber was coated with 10% Matrigel (356234, Corning, USA) when for invasion assay. The lower chamber was filled with fresh medium containing 10% FBS. After incubation of 24–48 h, the cells on the lower membrane were fixed with 4% paraformaldehyde and stained with crystal violet (C0121, Beyotime, China). Matrigel-invading or migratory cells were counted under a microscope.
Male BALB/c nude mice aged 4–5 weeks were purchased from HFK Bioscience (Beijing, China) and randomized into various groups, with 6–7 mice in each group. One million MHCC-LM3 cells with stable overexpressing VCP or 2.5 million Huh7 cells with stable silencing VCP (suspended in 0.1 mL PBS) were implanted subcutaneously under the right armpit of nude mice. Body weight and tumor size were measured every three days. The mice were sacrificed three weeks later or when they are moribund. Tumor volume (mm3) was calculated using the longest diameter × (shortest diameter)2 × 0.5. This experiment was performed in compliance with the requirements of the Department of Laboratory Animals, Xiangya Hospital, CSU, China.
For quantitative variables, a student’s t-test with one-tailed was used to compare the difference between two groups. When more than two groups are included in the experiments, the one-way ANOVA with a Brown-Forsythe test was accepted for multiple comparisons. The chi-square test was conducted for the qualitative variables. Kaplan–Meier method with a Log-rank test was operated to compare the survival distributions. All analyses were performed in GraphPad Prism software (version 8.0) and the results are presented as the mean ± SD or mean ± SEM. Differences were considered statistical significance at a P value < 0.05. Experimental data were obtained from three independent experiments unless otherwise presented. All *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns: no significance.