Cell lines and cell culture
The human HCC cell line HepG2 was obtained from the American Type Culture Collection (ATCC) and cultured in MEM (KeyGEN BioTECH, China) supplemented with 10% FBS (Gibco, USA) in a humidified incubator with 5% CO2 at 37 °C. The human normal liver cell line LO2 and the human HCC cell lines MHCC97L, MHCC97H, and HCCLM3 were obtained from the Cell Bank of the Chinese Academy of Sciences and cultured in DMEM (KeyGEN BioTECH) supplemented with 10% FBS in a humidified incubator with 5% CO2 at 37 °C. All cell lines were authenticated by the short tandem repeat (STR) profiling and tested for mycoplasma contamination. Exosome-depleted FBS was obtained by ultracentrifugation at 100,000 × g overnight at 4 °C. All cells ready for exosome isolation were cultured in medium supplemented with 10% exosome-depleted FBS in a humidified incubator with 5% CO2 at 37 °C.
Cell proliferation assay
Cells were seeded into 96-well plates (Jet Bio-Filtration, China) in triplicate at a density of 1000 cells per well. Cell viability was assessed by Cell Counting Kit-8 (CCK-8) assay (KeyGEN BioTECH) according to the manufacturer’s instructions. The optical density (OD) at 450 nm was measured at the indicated time points using a multifunctional microplate reader (Thermo Fisher Scientific, USA). Cell growth curves were generated using GraphPad Prism software (GraphPad Software, USA).
Colony formation assay
Cells were seeded into 6-well plates (Jet Bio-Filtration) in triplicate at a density of 2000 cells per well and cultured for 2 weeks. Subsequently, the cells were washed three times with PBS (HyClone, USA) and stained with crystal violet staining solution (KeyGEN BioTECH), followed by photoimaging under a microscope (Olympus, Japan). Colony formation rate = (number of colonies containing at least 50 cells/number of inoculated cells) × 100%.
Wound healing assay
Cells were seeded into 6-well plates in triplicate at a suitable density. When the cells grew to 80–90% confluence, wounded areas approximately 500-μm wide were created, and floating cells were removed. The cells were then cultured in serum-free medium for another 24 h. The process of cell migration to cover the wounded area was visualized and photoimaged under a microscope every 6 h. Data were analyzed using ImageJ software (NIH, USA).
Transwell migration and invasion assays
Cells were seeded into Transwell chambers (Corning, USA) in triplicate at a density of 5 × 104 cells per well in 200 μl of serum-free culture medium. For cell invasion assays, the Transwell membranes were precoated with Matrigel (BD, USA) before cell seeding. Then, 600 μl of culture medium supplemented with 20% FBS was added into the lower compartment of the Transwell chambers as a chemoattractant. The cells were allowed to migrate for 24 h or invade for 48 h and were then fixed with 4% paraformaldehyde (KeyGEN BioTECH) for 30 min and stained with crystal violet staining solution for 10 min. Crystal violet-stained cells adhering to the lower surface of the Transwell membranes were counted under a microscope in five random fields. Data were analyzed using ImageJ software.
Immunofluorescence (IF) staining
Cells were seeded in 6-well plates containing three cover slides at a density of 5000 cells per well and cultured overnight. The cells were then fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.2% Triton X-100 (Sigma, USA) for 5 min and blocked with 2% BSA (Sigma) for 1 h at room temperature. Subsequently, the cells were incubated with primary antibodies against E-cadherin (1:100, Proteintech, 20874-1-AP, USA), N-cadherin (1:100, Proteintech, 22018-1-AP), Vimentin (1:100, Proteintech, 10366-1-AP), integrin α6 (1:1000, Abcam, ab20142, UK) and integrin β4 (1:500, Abcam, ab133682) separately overnight at 4 °C. The next day, the cells were incubated with Alexa Fluor 488-conjugated secondary antibody (1:100, Proteintech, SA00013-1/ SA00013-2) or Alexa Fluor 594-conjugated secondary antibody (1:100, Proteintech, SA00013-3/SA00013-4) for 1 h at room temperature in the dark. Cell nuclei were counterstained with DAPI (Sigma) for 5 min at room temperature in the dark. IF signals were visualized using an Olympus IX81 fluorescence microscope (Olympus) and a Leica TCS SP5II confocal microscope (Leica, Germany). Data were analyzed using ImageJ software.
SDS-PAGE and western blot analysis
Total cellular and exosomal proteins were obtained using a RIPA lysis buffer (KeyGEN BioTECH) containing a protease inhibitor cocktail (Sigma) and a phosphatase inhibitor cocktail (Sigma) for use in further assays. A bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific) was used to measure protein concentrations. Equal amounts of cell lysate or exosome lysate were resuspended in 5× loading buffer (KeyGEN BioTECH) and were subsequently incubated at 95 °C for 5 min and then centrifuged in a microcentrifuge at 15,000 × g for 5 min. Samples were separated on 10% SDS-PAGE gels (KeyGEN BioTECH) and transferred to nitrocellulose membranes (Millipore, USA). Membranes were stained with Ponceau red staining solution (KeyGEN BioTECH) and were then washed with TBS containing 0.1% Tween 20 (TBS-T). The destained membranes were blocked with 5% nonfat milk (BD) for 1 h at room temperature. Then, primary antibodies against E-cadherin (1:5000, Proteintech, 20874-1-AP), N-cadherin (1:2000, Proteintech, 22018-1-AP), Vimentin (1:2000, Proteintech, 10366-1-AP), MMP2 (1:1000, Abcam, ab92536), MMP9 (1:1000, Abcam, ab76003), GM130 (1:1000, Abcam, ab52649), GRP94 (1:1000, Abcam, ab238126), ALIX (1:10,000, Abcam, ab186429), TSG101 (1:1000, Abcam, ab125011), CD63 (1:1000, Abcam, ab134045), CD81 (1:1000, Abcam, ab109201), ENO1 (1:1000, Abcam, ab155102), HA (1:4000, Abcam, ab9110), integrin α1 (1:500, Proteintech, 22146-1-AP), integrin α2 (1:10,000, Abcam, ab133557), integrin α3 (1:500, Proteintech, 66070-1-Ig), integrin α5 (1:1000, Abcam, ab150361), integrin α6 (1:2000, Abcam, ab181551), integrin αV (1:5,000, Abcam, ab179475), integrin β1 (1:1000, Abcam, ab52971), integrin β3 (1:1000, Abcam, ab119992), integrin β4 (1:1000, Abcam, ab182120), integrin β6 (1:10,000, Abcam, ab187155), FAK (1:2000, Abcam, ab40794), FAK (Y397) (1:1000, Abcam, ab81298), FAK (Y576 + Y577) (1:50,000, Abcam, ab76244), FAK (Y861) (1:10,000, Abcam, ab81293), FAK (Y925) (1:1000, Abcam, ab230813), Src (1:10,000, Abcam, ab109381), Src (Y418) (1:1000, Abcam, ab40660), Src (Y419) (1:5000, Abcam, ab185617), Src (Y529) (1:5000, Abcam, ab32078), p38MAPK (1:1000, Abcam, ab170099), p38MAPK (T180 + Y182) (1:1000, Abcam, ab195049), GAPDH (1:10,000, Abbkine, A01020, USA), and β-tubulin (1:10,000, Abbkine, A01030) were diluted in primary antibody diluent (KeyGEN BioTECH) and incubated with membranes overnight at 4 °C. The next day, the membranes were washed three times with TBS-T and were then incubated with secondary antibody (1:10,000, Abbkine, A21020) for 1 h at room temperature. Membranes were washed three times with TBS-T before scanning and visualization of immunoreactions using the ECL western blot substrate (NCM Biotech, China). Protein expression of three independent experiments was quantified using ImageJ software.
Exosome isolation, characterization, and analyses
Exosomes were isolated for mass spectrometry using an Exosome Isolation Kit (PTM BIO, China). Exosomes used for all other experiments were isolated by ultracentrifugation. The indicated cells at 70–80% confluence were cultured in medium supplemented with 10% exosome-depleted FBS for 72 h. Supernatant fractions collected from 72-h cell cultures were centrifuged at 300 × g for 10 min, 2000 × g for 20 min, and 10,000 × g for 30 min. The supernatant fractions were then filtered through a 0.22-µm filter. Exosomes were collected by ultracentrifugation at 100,000 × g for 70 min at 4 °C. The exosome pellet was resuspended in 10 ml of PBS and purified by ultracentrifugation at 100,000 × g for 70 min at 4 °C (Beckman, USA). The purified exosomes were resuspended in PBS for subsequent experiments. The exosomal protein concentration was measured by using a BCA Protein Assay Kit. Exosome preparation was verified by western blot analysis and electron microscopy. Purified exosomes were dropped onto electron microscopy grids, allowed to absorb for 10 min, and then negatively stained with 2% phosphotungstic acid for 5 min. The electron microscopy grids were observed under a transmission electron microscope (JEOL, Japan) at 120 kV. Exosome sizes and particle numbers were assessed using a Zeta View PMX110 (Particle Metrix, Germany) equipped with a blue laser (405 nm) and analyzed using nanoparticle tracking analysis (NTA) software (Zeta View 8.02.28).
Exosome labeling and tracing
Purified exosomes were fluorescently labeled using a PKH67 Green Fluorescent Cell Linker Kit (Sigma) according to the manufacturer’s protocol. Labeled exosomes were washed in 10 ml of PBS and recollected by ultracentrifugation. Purified exosomes labeled with PKH67 were resuspended in serum-free medium and then used for culture with cells for 2 h. Subsequently, cells were fixed in 4% paraformaldehyde for 30 min, and cell nuclei were counterstained with DAPI for 5 min at room temperature in the dark. Fluorescence signals were visualized using a Leica TCS SP5II confocal microscope.
The 3- to 4-week-old female BALB/c-nu/nu nude mice were randomized. Approximately 1 × 106 cells were suspended in 30 μl of PBS/Matrigel (1:2) cocktail or 30 μl of exosome/Matrigel (1:2) cocktail containing 5 μg of exosomes. Through a 1-cm transverse incision in the upper abdomen, they were orthotopically injected in the hepatic lobe of each nude mouse anesthetized with pentobarbital sodium. Another approximately 1 × 106 cells were suspended in 100 μl of PBS or 100 μl of exosomes (50 μg/ml) and were injected intravenously into each nude mouse. After 4 or 8 weeks, the nude mice were euthanized, and their livers and lungs were harvested and analyzed to assess HCC cell proliferation and metastasis in vivo. All nude mice were purchased by and raised in the Experimental Animal Center of Dalian Medical University. All nude mice were raised in a specific pathogen-free (SPF) environment and were provided sterilized feed and water. All animal experiments were approved by the Experimental Animal Ethics Committee of Dalian Medical University. All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health.
Establishment of stable overexpression and knockdown cell lines
For knockdown of ENO1 in HCC cell lines, shRNA sequences targeting ENO1 (shENO1-1: 5′-GGACTTCAAGTCTCCCGATGA-3′, shENO1-2: 5′-GCGGTTCTCATGCTGGCAACA-3′, shENO1-3: 5′-GCTCAAAGTCAACCAGATTGG-3′) were inserted into the LV3 lentiviral vector and were then packaged in 293T cells using RNAi-Mate (GenePharma, China). The shENO1 vector (LV3-shENO1) or mock vector (LV3-NC) was transfected into HCCLM3 cells. For overexpression of ENO1 in HCC cell lines, the full-length cDNA encoding ENO1 was inserted into the LV5 lentiviral vector and was then packaged in 293T cells using RNAi-Mate. The ENO1 vector (LV5-ENO1) or mock vector (LV5-NC) was transfected into MHCC97L and HepG2 cells. Transfected cells used for overexpression or knockdown were selected with puromycin (5 μg/ml) for 2 weeks. The efficiency of stable overexpression or knockdown was evaluated by western blotting.
The exosome pellet was washed with PBS and sonicated three times on ice using a high-intensity ultrasonic processor (Scientz, China) in 4 volumes of lysis buffer (8 M urea, 1% protease inhibitor cocktail). The remaining debris was removed by centrifugation at 12,000 × g for 10 min at 4 °C. Finally, the supernatant was collected, and the protein concentration was measured with a BCA protein assay kit according to the manufacturer’s instructions. For digestion, the proteins in solution were reduced with 5 mM dithiothreitol for 30 min at 56 °C and alkylated with 11 mM iodoacetamide for 15 min at room temperature in the dark. The protein sample was then diluted by adding 100 mM tetraethylammonium bromide (TEAB) to a urea concentration of less than 2 M. Finally, trypsin was added at a 1:50 trypsin:protein mass ratio for the first digestion overnight and a 1:100 trypsin:protein mass ratio for a second digestion for 4 h. After trypsin digestion, peptides were desalted on a Strata X C18 SPE column (Phenomenex) and dried in a vacuum. Peptides were reconstituted in 0.5 M TEAB and processed according to the manufacturer’s protocol for the tandem mass tag (TMT) kit. Tryptic peptides were fractionated via high-pH reverse-phase HPLC using an Agilent 300Extend-C18 column (5-μm particles, 4.6-mm inner diameter (ID), 250-mm length). Tryptic peptides were dissolved in solvent A (0.1% formic acid) and loaded directly onto an in-house-constructed reversed-phase analytical column (15-cm length, 75-μm ID). The gradient was composed of an increase from 6 to 20% solvent B (0.1% formic acid in 90% acetonitrile) over 20 min, from 20 to 35% solvent B over 13 min, and from 35 to 80% over 3 min, followed by a final holding step at 80% for 3 min. All steps were performed at a constant flow rate of 300 nL/min on an EASY-nLC 1000 UPLC system. Peptides were subjected to nanospray ionization (NSI) tandem mass spectrometry (MS/MS) in a Q ExactiveTM Plus (Thermo Fisher Scientific) coupled online to the UPLC system. The applied electrospray voltage was 2.0 kV. The m/z scan range was 400–1500 for a full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using a normalized collision energy (NCE) setting of 100, and fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure alternating between one MS scan and 20 subsequent MS/MS scans with a dynamic exclusion window of 15.0 s was used. The automatic gain control (AGC) was set at 5 × 104. The resulting MS/MS data were processed using the MaxQuant search engine (v.220.127.116.11). Tandem mass spectra were searched against the SwissProt Human database concatenated with the reverse decoy database. Trypsin/P was specified as the cleavage enzyme, allowing up to 2 missed cleavages. The mass tolerance for precursor ions was set at 20 ppm in the first search and 5 ppm in the main search, and the mass tolerance for fragment ions was set at 0.02 Da. The false discovery rate (FDR) was adjusted to 1%.
Immunohistochemistry (IHC) staining and scoring
After deparaffinization with xylene, rehydration in an ethanol gradient, heat-mediated antigen retrieval in citric acid and immersion in 3% hydrogen peroxide, tissue sections were incubated with the antibody against ENO1 (1:100, Abcam, ab155955) overnight at 4 °C. The next day, tissue sections were incubated with an HRP-conjugated secondary antibody (1:1000, Abcam, ab6721) for 1 h at room temperature. Tissue sections were developed with DAB solution and examined under a microscope. Tissue sections were counterstained with hematoxylin. The ENO1 expression score was assessed according to both the proportion (negative, scored 0; 1–25%, scored 1; 25–50%, scored 2; 50–75%, scored 3; and 75–100%, scored 4) and intensity (negative, scored 0; 1+, scored 1; 2+, scored 2; 3+, scored 3) of positively stained cells. Finally, the product of the “staining intensity score” and “positive staining rate score” was used as the total score for grouping. According to the data distribution, sections with a total score of 4 or less were considered the low ENO1 expression group, and sections with a total score of greater than 4 were considered the high ENO1 expression group. These scores were determined independently by two experienced pathologists in a blinded manner, and the mean percentage values were used.
Data are expressed as the mean ± standard deviations (SD) and analyzed by GraphPad Prism software. The data of different experimental groups meet normal distribution and the variance is similar between the groups. Statistical significance was analyzed by two-tailed Student’s t-test or ANOVA where applicable. The correlation of ENO1 expression with clinicopathological parameters in HCC patients was evaluated by the chi-square test and Fisher’s exact probability method. Survival curves were constructed via the Kaplan–Meier method and analyzed by the log-rank test. Univariate and multivariate analyses based on the Cox proportional hazards regression model were performed to identify the factors with a significant influence on survival. P < 0.05 was considered statistically significant.