Primary HCC tumor samples, paired tumor adjacent tissues and serum samples of 49 HCC patients and serum samples of 28 healthy individuals were obtained from Tianjin First Central Hospital. Among them, serum samples from six HCC patients and two healthy individuals were subjected to miRNome sequencing. Serum samples and surgically removed tissues were frozen and transported in liquid nitrogen. All of the samples were obtained with written informed consent from patients and approval from the Ethics Committee of Tianjin First Central Hospital and Medical School of Nankai University.
Adult male BALB/c and NOD-SCID mice were ordered form HuaFuKang Bioscience Co. (Beijing, China), housed under specific pathogen free environmental conditions. Mice were intraperitoneal injectied with 4% chloral hydrate (100 μl per 10 g mouse body weight) before surgery, and were sacrificed via intraperitoneal injection of fatal dose of 4% chloral hydrate after study.
Exosome extraction from serum and cell supernatant samples using the exosome isolation reagent (Ribobio Co., China) were performed according to the manufacturer’s protocol. For electron microscopy, the serum was pre-cleared by centrifugation at 300 × g for 5 min, 2000 ×g for 10 min, and then at 10000 × g for 60 min. Exosomes were isolated by ultracentrifugation at 100000 ×g for 130 min, followed by PBS washing under the same ultracentrifugation conditions. Exosomes were resuspended in 100 μl PBS, fixed with 2% paraformaldehyde, loaded on 200-mesh Formvar-coated grids, contrasted, and embedded for imaging.
Cell lines and reagents
Human umbilical vein endothelial cells (HUVECs) and human HCC cell lines Hep3B and HepG2 were obtained from ATCC. HCC cell line SK-Hep-1, SMMC-7721, PLC/PRF/5, and normal human hepatocyte cell line L-02 were purchased from the Chinese Academy of Sciences (Shanghai, China). Hep3B, HepG2, SK-Hep-1 and PLC/PRF/5 cells were cultured in MEM containing 10% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, and 100 μg/ml non-essential amino acids. SMMC-7721 cells were grown in DMEM containing 10% FBS and 100 U/ml penicillin/streptomycin. L-02 and HUVECs were cultured in RPMI-1640 containing 10% FBS and 100 U/ml penicillin/streptomycin. MiR-1290 mimics and miR-1290 mimic-FAM were purchased from Ribobio Co.
Cell viability assays
For cell viability assays, HUVECs were seeded in 96-well plates in 100 μl of medium, followed by transfection with 50 nM miR-1290 mimics. Five parallel wells were assigned for each group. At 0, 24, 36, 48, and 60 h after transfection, CCK-8 solution (10 μl) was added to each well, and absorbance values were measured at 450 nm after incubation for 1.5 h at 37 °C.
RNA extraction and quantitative real-time PCR (qRT-PCR)
Tissues were grinded using a homogenizer in Trizol reagent (Invitrogen, USA). Cells were collected, washed and lysed in Trizol reagent, and total RNA was isolated following the manufacturer’s instructions. qRT-PCR was performed using a standard SYBR-Green PCR kit protocol. Primers are listed in Table S1.
Western blotting was performed in accordance with a previous protocol.20 Briefly, proteins were loaded on 5–12% tris-acrylamide gels and membranes were blotted with specific antibodies. The primary antibodies used in this study were SMEK1 (ab70635, Abcam), pVEGFR2 (#4991, Cell Signaling Technology), and VEGFR2 (#9698, Cell Signaling Technology) at a dilution of 1:1000. Bands were detected using a Gel imaging system (SYNGENE).
Plasmid construction and transfection
Human SMEK1 cDNA was cloned from HUVECs and ligated into pLV-EF1α-MCS-IRES-Bsd vector (Biosettia Inc.). SMMC-7721, Hep3B, and HUVECs (3 × 105 cells/well) were seeded in 6-well plates, incubated overnight, and then transfected with miR-1290 mimics using Lipofectamine2000 (Invitrogen) and Opti-MEM (Corning) according to the manufacturer’s instruction. Primers are listed in Table S2.
Cell migration assays
Cell migration was evaluated by performing wound-healing and transwell assays. For wound-healing assay, cells were seeded in 6-well plates at 2 × 105 cells per well, and after 48 h transfection, the cell monolayer was scraped using a 10 μl tip. The initial gap length and the residual gap length after wounding were calculated based on photomicrographs using ImageJ software. For transwell assay, after transfection for 24 h, cells were plated in 24-well plates at 5 × 104 in the upper 8-μm chambers (BD bioscience). Medium containing 10% FBS in the lower well and 2% FBS in the upper chamber served as the chemoattractant. After another 16 h incubation, cells on the upper surface of the membrane were scraped off and those on the bottom were stained with crystal violet. These cells were photographed using an optical microscope.
Tube formation and Matrigel plug assays
For tube formation assay, a pre-chilled 48-well plate was coated with 150 μl of Matrigel (BD bioscience) and incubated at 37 °C for 30 min. HUVECs (3 × 104 cells/well) were transfected with miR-1290 and then seeded on this plate. After 5 h, photographs were taken, and the tubes were counted.
For in vivo Matrigel plug assay, male BALB/c mice were administered a subcutaneous injection of 750 μl of mixture containing 500 μl Matrigel and 250 μl EBM2 medium with or without miR-1290 (3 mice for each group). After 10 days, the Matrigel plugs were imaged and snap frozen in the presence of optimum cutting temperature (OCT) medium before sectioning. Frozen Matrigel sections (8 μm) were fixed in cold methanol and immunostained with a CD31 antibody (ab28364, Abcam, USA).
Slides were blocked with 2% BSA and then incubated with an anti-CD31 antibody (ab28364, Abcam) for 1 h. After fluorophore-conjugated secondary antibody incubating and PBS washing, slides were incubated with DAPI (Invitrogen) for 2 min. Images were obtained using a microscope (Olympus IX73).
Dual luciferase reporter assay
HUVECs were cultured in 24-well plates at a density of 2 × 105 cells/well overnight, which was followed by co-transfection with miR-1290 mimics, pmirGLO, and the pRL-TK plasmid. After 36 h of transfection, the inhibition of miR-1290 was quantified as the ratio of firefly luciferase activity to Renilla luciferase activity in each well.
Male NOD-SCID mice (6-weeks-old) were separated randomly into two groups (n = 6 each), and 2 × 106 SMMC-7721 cells were inoculated subcutaneously into each mouse, and tumors were allowed to grow for 10 days. Tumors were peritumorally treated with miR-1290 antagomir or miRNA antagomir negative control (NC), and tumor volume was measured with calipers once every 3 days. All mouse experiments were conducted in accordance with the standard operating procedures approved by the Institute Research Ethics Committee at the Nankai University.
IHC staining was performed using paraffin-embedded human HCC tissues and mouse xenografts tumors. These tissues were probed separately with an antibody against Ki67 (ab16667, Abcam), cleaved-caspase 3 (#9664, Cell Signaling Technology), and CD31 (ab28364, Abcam) at a 1:100 dilution.
Statistical analyses were performed using SPSS 23.0 software; the data from all experiments are presented as means ± SD and represent three independent experiments. A paired t-test was used to compare gene expression in tumor and adjacent non-tumor tissue samples. Where appropriate, a Student’s t-test for unpaired observations was applied. A value of P < 0.05 was considered significant.