Patients and tissue samples
Ninety pairs of primary HCC tissue and adjacent non-tumor liver tissue (> 2 cm and < 5 cm from tumor edge) were collected from patients (56 males and 41 females; age, 41-73 years; mean age, 52.6 years) who underwent surgical resection at our hospital from October 2013 to January 2015. The tissue samples were immediately frozen and preserved at -800C in liquid nitrogen. None of the patients suffered from hepatitis or had previously accepted anti-inflammatory treatment or oncotherapy.
Written informed consent was obtained from all patients before the study and the diagnosis of all tissue samples was made and independently confirmed by two pathologists. Overall survival was calculated during the duration between surgery and death or last follow-up. This study was approved by the Shenzhen Hospital, Peking University Institutional Ethics Committee and conducted following Ethical Principles for Medical Research Involving Human Subjects of the Helsinki Declaration.
HCC cell lines (HL7702, HCCLM3, HepG2, and MHCC97-H) and human immortalized hepatocyte line L02, and human BM-MSCs were obtained from the American Type Culture Collection. The cells were all cultured with DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin.
Flow cytometry identification of the BM-MSCs
Specific stem cell markers were detected using flow cytometry, as previously described 24, 25. In brief, BM-MSCs were harvested and washed. Then, Phycoerythrin (PE)-labeled anti-CD34, CD44, CD45, CD90, and the isotype control was incubated with the BM-MSCs for 20 min. After washing with PBS, the BM-MSCs were examined using flow cytometry.
Isolation and identification of the BM-MSC-derived exosomes
Exosomes were extracted from the culture supernatant of the BM-MSCs using ultracentrifugation and their identity was confirmed using previously reported methods 26, 27. In brief, the BM-MSCs were cultured in DMEM containing 10% FBS (FBS was pre-centrifuged at 16,000 ×g for 60 min to remove the exosomes). After 24 h of culture, the medium supernatant was centrifuged at 16,000 ×g for 60 min at 4°C and the exosomes were harvested.
The cells were plated and cultured at 37°C for 24 h. Next, the cells were transfected with miR-424-5p mimic, miR-424-5p inhibitor, siRNA-FOXK1, or the NC, respectively, using a Lipofectamine 2000 system, following the manufacturer’s instructions. Transfection efficiency was measured after 48 h.
For exosome labeling, a PKH26 kit was used as instructed by the manufacturer. In brief, exosomes were suspended in Diluent C and then mixed with PKH26. After incubation for 20 minutes in the dark at room temperature, the labeling reaction was stopped and the labeled exosomes were ultra-centrifuged at 100,000 ×g for 60 min. After washing with PBS, the exosomes were again ultra-centrifuged at 100,000 ×g for 60 min and resuspended in a serum-free medium, and co-cultured with the HCC cells. Then, the labeled exosomes were observed under a confocal laser scanning microscope.
Transmission electron microscopy
The isolated exosomes were fixed in 200 μL of 3% glutaraldehyde for 100 min. A drop (20 μL) of exosomes was transferred onto a formvar-carbon-coated grid. After rinsing, the grids were stained with 1% uranyl acetate solution for 5 min. Then, the grids were washed twice and examined under a transmission electron microscope at 80 kV.
Exosome uptake experiment
MHCC97-H cells were incubated with the BM-MSC-derived exosomes and stained with 1 mL of 4’,6-diamidino-2-phenylindole 2 HCI for 10 min. BM-MSC uptake by the MHCC97-H cells was observed under a fluorescence microscope.
Total RNA was extracted from the tissues and cells using TRIzol reagent, and reverse transcription was performed using a TaqMan microRNA Reverse Transcription Kit. PCR amplification was conducted using SYBR Premix Ex Taq with U6 and GAPDH as the loading controls of miR-424-5p and FOXK1.
Dual-luciferase reporter gene assay
The binding site between miR-424-5p and FOXK1 was predicted by Jefferson. The wild type (Wt) and mutant type (Mut) FOXK1 3’UTR luciferase vectors (WT-3’UTR and MUT-3’UTR) were constructed and cloned into the pmiR-RB-reporter plasmid. Then, Pmir-FOXK1-WT and Pmir-FOXK1-MUT were transfected into HL-7702 cells with miR-424-5p mimic, miR-424-5p inhibitor, and miR-424-5p-NC using a Lipofectamine 2000 system for 48 h. Finally, the luciferase activity of all transfected cells was detected.
Western blotting analysis
Total protein in the cells and exosomes was extracted, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was conducted, and then the samples were transferred onto membranes. The membranes were blocked with 5% skim milk powder and incubated with primary antibodies FOXK1 (1: 1000; Abcam, MA, USA), CD63 (1: 500), and CD81 (1: 500) at 4℃ overnight, and then with the respective secondary antibody (1: 3000, CA, USA). An enhanced chemiluminescent system was used for detection using GAPDH as the internal reference.
Cell apoptosis and viability analyses
Apoptotic BM-MSCs were detected using FCM with an Annexin-V FITC kit, according to the manufacturer’s instructions. The apoptosis of the HCC cells was determined using flow cytometry, and an FC500 MCL flow cytometer was used to detect the apoptosis rate. Cell activity was analyzed using an MTT assay, as previously described and absorbance was assessed at 570 nm using a microplate reader (Bio-Rad Laboratories, CA, USA).
The transfected cells were added into Transwell apical chambers coated with Matrigel (not used in the migration assay), while the basolateral chambers were appended with 500 μL of complete medium. After 24 h of incubation, the cells on the microporous membrane were removed and the cells in the chambers were fixed and stained with crystal violet for 10 min, and were then observed under an inverted microscope.
Subcutaneous in vivo tumorigenesis in nude mice
Male BALB/c nude mice were subcutaneously injected with transfected HL-7702 cells. After tumors had developed, the tumors were measured and volume was calculated using the formula: ab2/2 (a: length diameter, b: width diameter). The mice were euthanized on the 35th day and the tumors were isolated and weighed. Animal experiments were conducted in strict accordance with the Guide to the Management and Use of Laboratory Animals issued by the National Institutes of Health. The mice were housed as stipulated by the protocols of animal experiments and were approved by the Institutional Animal Care and Use Committee of our Hospital.
All data analyses were conducted using SPSS 21.0 software. Measurement data that conformed to normal distribution were expressed as mean ± standard deviation. The unpaired t-test was conducted for comparisons between two groups, while one-way analysis of variance (ANOVA) was used for comparisons among multiple groups and Tukey’s post hoc test was used for pairwise comparisons after one-way ANOVA. A P value of < 0.05 was indicative of a statistically significant difference.