Cell lines and culture
The HCC cell lines Huh-7 and SMMC-7721 were used in this study. The cell lines were provided and identified by our laboratory of living donor liver transplantation. The cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 µg/mL streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C in 5% CO2. RPMI-1640 (DMEM; Gibco) containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin was used for THP-1 cells. For M2 polarized macrophages, THP-1 cells were treated with 5 ng/mL PMA (Sigma, USA) for 24 h and then incubated with 20 ng/mL IL-4 plus 20 ng/mL IL-13 (PeproTech, USA) for 48 h. M2 polarized macrophages were identified by microscopy, flow cytometry, and immunofluorescence.
Isolation of macrophage exosomes
M2 macrophages obtained from THP-1 were incubated with 10% exosome-free FBS for 48 h. Then, the medium was collected and centrifuged at 1,000g for 5 min at room temperature and then at 15,000g for 15 min at 4°C, followed by filtration with 0.22-µm membrane to remove cells, cell fragments, and organelles. The supernatant was then centrifuged at 110,000g for 70 min at 4°C. The precipitate was then collected and resuspended in PBS. The exosomes were characterized by transmission electron microscopy and western blotting.
Transmission electron microscopy (TEM)
For TEM, the exosomes were resuspended in PBS, dropped onto a copper mesh, and stained with 1% uranyl acetate. The mesh was dried for 5 min and observed under a transmission electron microscope (FEI, Tecnai G2 spirit).
The extracted exosomes were incubated with 0.5 mL of diluent C. Approximately 4UL of a PKH67 dye solution were incubated with 0.5 mL of diluent C. The exosomes and PKH67 were mixed and incubated overnight at 4°C. The mixture was rinsed with PBS to remove excess dyes. The prepared dye solution was seeded to the prepared cells for 24 h of incubation. Then, images of the cells were captured under a laser confocal microscope.
For the lentivirus (Genepharma, Shanghai, China), cells were plated into six-well plates at a density of 2×105 cells per well. When the density reached 30–40% after incubation, the lentiviruses were transfected into the HCC cells. When cell confluency reached 80–90%, the cells were transferred into 100-mm dishes and selected for 2 weeks using puromycin (10 µg/mL). For shRNA, Lipofectamine 3000 (Invitrogen) was used according to the manufacturer's protocol. Briefly, the cells were plated in six-well plates at a density of 1.5×105 cells per well one day before transfection. When the cells were in the log phase, a mimic was added to each well. The medium was replaced after incubation for 6 h. The cells were harvested 48–72 h later for analysis.
RNA isolation and quantitative real-time PCR
Total RNA was extracted from human specimens and cells using TRIzol reagent (Takara, Dalian, China) according to the manufacturer’s instructions. RNA quality and concentration were assessed using a Nanodrop 2000 system (NanoDrop Technologies, IL, USA). The cDNAs of miRNA were reverse transcribed using the reverse translate kit PrimeScript RT Master Mix (DRR037A; Takara) according to the manufacturer’s protocol. Sequence-specific primers for U6 and miR-23a-3p were synthesized by RiboBio (Guangzhou, China). Real-time polymerase chain reaction was performed with SYBR Premix ExTaq (TaKaRa, Dalian, China) with the ABI Prism 7900HT (Applied Biosystems, Foster City, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal control for mRNA quantification. The relative expression was calculated using the 2–ΔΔCT method. The primer sequences used in real-time PCR were displayed in Supplementary Table S1.
Cells and tissue specimens were lysed using a lysis buffer (10 mM Tris, 1 mM EDTA, 1 mM DTT, 60 mM KCl, 0.5% v/v NP-40, and protease inhibitors) on ice. Protein samples (20 µg) were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride membrane. After blocking, the bands were incubated with specific antibodies. Proteins were visualized with an enhanced chemiluminescence detection kit according to the manufacturer’s recommendations (Beyotime, Shanghai, China). Relative protein levels were calculated based on GAPDH levels. All antibodies used in the study was displayed in Supplementary Table S2
A diaminobenzidine detection kit (Maixin-Bio, Fuzhou, China) was used for immunohistochemical staining according to the manufacturer’s instructions. The dissected HCC and peritumor tissues were fixed and embedded in paraffin. Then, 5-µm thick consecutive sections were cut and mounted on glass slides, dewaxed, rehydrated, and antigen retrieved. The sections were incubated with a primary antibody at 37°C for 1 h, a biotin-labeled secondary antibody for 10 min, and a streptavidin–peroxidase conjugate for 10 min. A 0.02% diaminobenzidine solution was used as chromogen to visualize peroxidase activity. The sections were lightly counterstained with hematoxylin, mounted with Permount, and examined with light microscopy.
Migration and invasion analysis was performed using Transwell® chambers (Corning Incorporated, Corning, NY, USA). For migration, following suspension in serum-free medium, 3×104 cells were seeded into the Transwell® insert supplemented with DMEM containing 5% serum. After 48 h of incubation, the cells on the bottom side of the membrane were fixed with 95% alcohol and stained with crystal violet for 20 min at room temperature. Then, the number of cells on the lower side of the filter was counted under a microscope. Each experiment was performed in triplicate. For HUVEC cell migration, 1×104 cells were seeded and then incubated 24 h. For invasion, Matrigel® was plated in the upper surface before cell inoculation. The other steps were the same as in the migration assay.
Endothelial permeability assay in vitro
Endothelial permeability assay was performed according to a previous study16. Briefly, 2 × 104 of HUVEC cells were seeded into 24-well Transwell® filters (8-µm pore size; Corning) and then incubated when cell confluency reached 100%. Then, the 24-well Transwell® filters were carefully washed with PBS. Then, 20 mg/mL rhodamine-dextran (average MW: ~70,000; Sigma-Aldrich) was added into the upper chamber and after 1 h of incubation, the lower medium was collected and assessed under laser confocal scanning microscopy.
Transendothelial invasion assay
A transendothelial invasion assay was performed according to a previous study17. Endothelial monolayers were prepared as earlier described. The 24-well Transwell® filters were placed into new 24-well plates, and then 5×104 of GFP-positive HCC cells were seeded into the upper chamber, and the lower chamber was filled with 750 µL of 10% exosome-depleted FBS-SFM. After a 12-h incubation, the cells in the upper and lower chambers were collected and counted under a fluorescent microscope.
Tube formation assay
Growth factor-reduced Matrigel® (BD Biosciences, San Jose, CA, USA) was diluted with serum-free medium at a 1:1 ratio and then 300 µL were placed into each well of 24-well plates. The plates were incubated for 30 min. The different group of HUVEC cells were suspended at a density of 1.2×106, and 100 µL of the cell suspensions were seeded into the 24-well plates. After 4–6 h of incubation, tube images were captured using a digital camera. Tube formation was determined using ImageJ software.
For immunofluorescence, cells were fixed with 4% formaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 1% bovine serum albumin in PBS. Cells were incubated with the primary antibody overnight at 4°C. The cells were then incubated with FITC-conjugated goat anti-rabbit IgG after three washes. Finally, the cells were washed and mounted with a mounting medium containing 4,6-diamidino-2-phenylindole (DAPI). Images were captured with a scanning microscope.
Briefly, the cell culture supernatant was collected, centrifuged at 1,000g at 4°C for 30 min, and the supernatant was stored at -80°C until use. Samples to be tested were thawed on ice. Standard wells were set up, and then 50 µL of pre-replaced standards at different concentrations were added to each well. Then, 50 µL of each sample to be tested were added into each well. Later, 50 µL of horseradish were added per well and incubated for 1 h at 37°C. The liquid was later discarded and then 300 µL of cleaning solution were added. After 2 min, the cleaning solution was discarded, and the microwell plate was patted dry on the absorbent paper to remove any residual liquid. The microwell plate was rinsed for another 5 times, and then 50 µL of the substrate were added to each well and incubated for 15 min. Then, 50 µL of a stop solution was added to terminate the reaction, and then the absorbance of each well was assessed at a wavelength of 450 nm.
Briefly, cells were plated into 96-well plates at a density of 3,000 cells/well. In each well, 10 µL of CCK-8 were added at various time points (0, 24, 48, and 72 h), and the cells were incubated at 37°C for another 2 h. The absorbance at a wavelength of 450 nm was measured and used to generate cell growth curves.
RIP assays were performed using an RIP RNA-binding protein immunoprecipitation kit (Millipore, USA) according to the instruction. Briefly, the cells were lysed on ice-cold lysates supplemented with protease inhibitors, RNase inhibitors, and 1 mM PMSF and centrifuged at 1,500 rpm at 4°C for 15 min. The protein extract (1 mg) was incubated with 3µg of rabbit anti hnRNPA1 antibody (Cell Signaling Technology, USA) or rabbit IgG (Proteintech, USA) in reverse rotation at 4°C overnight. Then, about 30µL A/G protein beads were added, and the cells were suspended and incubated at 4°C for 4 h. Then, the beads were washed five times.Co-immunoprecipitated miRNA was extracted by Ambion. The separated RNA was analyzed.
MiRNA targets were predicted using the TargetScan and miRDB. Cells (5 ×104) were seeded into 24-well plates and cultured for 24 h. The reporter luciferase plasmid (100 ng), pGL3, pGL3--mut, or control luciferase plasmid, and 5 ng pRL-TK Renilla plasmid (Promega, Madison, WI, USA) was transfected into the cells using Lipofectamine 3000 according to the manufacturer’s instructions. Luciferase and Renilla signals were measured 48 h after transfection using a Dual-Luciferase Reporter Assay Kit (Promega) according to the manufacturer’s protocol.
Statistical analyses were performed using Prism 5.0 (GraphPad Software, La Jolla, CA, USA) software. The results are presented as the mean ± standard error of the mean (SEM). The Student's t test was used for assessment of group differences. Differences with P < 0.05 were considered statistically significant.