Patients and specimens
Primary tumor tissues and their corresponding adjacent non-tumorous liver specimens were obtained from 84 patients who were diagnosed with HCC during hepatic resection in the First Affiliated Hospital of China Medical University between July 2013 and July 2014. None had received preoperative radiotherapy or chemotherapy before surgery. Fresh specimens were snap-frozen and stored in liquid nitrogen tanks immediately after resection. The clinical and pathological parameters are shown in Table
1. This study protocol was approved by the Institutional Ethics Committee of China Medical University, and human tumor tissues for this research were obtained with informed consent.
Table 1
miR-655-3p expression and clinicopathological features in hepatocellular carcinoma patients
Age (years) |
≥ 53 | 43 | 27(62.8 %) | 16(37.2 %) | 0.69 |
< 53 | 41 | 24(58.5 %) | 17(41.5 %) | |
Gender |
Male | 71 | 45(63.4 %) | 26(36.6 %) | 0.242 |
Female | 13 | 6(46.2 %) | 7(53.8 %) | |
HBsAg status |
Positive | 62 | 36(58.1 %) | 26(41.9 %) | 0.404 |
Negative | 22 | 15(68.2 %) | 7(31.8 %) | |
AFP (ng/ml) |
≥ 20 | 48 | 30(62.5 %) | 18(37.5 %) | 0.699 |
< 20 | 36 | 21(58.3 %) | 15(41.7 %) | |
Liver cirrhosis |
Yes | 63 | 38(60.3 %) | 25(49.7 %) | 0.897 |
No | 21 | 13(61.9 %) | 8(39.1 %) | |
Tumour size (cm) |
< 5 | 44 | 22(50 %) | 22(50 %) | 0.035* |
≥ 5 | 40 | 29(72.5 %) | 11(27.5 %) | |
PVTT |
Yes | 9 | 9(100.0 %) | 0(0.0 %) | 0.028* |
No | 75 | 42(56 %) | 33(44 %) | |
TNM Classification |
I + II | 50 | 24(48 %) | 26(52 %) | 0.004** |
III + IV | 34 | 27(79.4 %) | 7(20.6 %) | |
Tumour differentiation |
Well | 37 | 21(56.8 %) | 16(43.2 %) | 0.721 |
Moderate | 34 | 21(61.8 %) | 13(48.2 %) | |
Poor | 13 | 9(69.2 %) | 4(30.8 %) | |
Metastasis |
Yes | 33 | 27(81.8 %) | 6(18.2 %) | 0.001** |
No | 51 | 24(47.1 %) | 27(52.9 %) | |
HCC cell lines and cell cultures
Seven HCC cell lines, HepG2, SK-hep1, HCCLM3, Huh7, MHCC-97H, MHCC-97 L, BEL-7402, and one normal liver cell line, LO2, were used in this study. The Huh7, BEL-7402, SK-hep1, HCCLM3 and LO2 human cell lines were obtained from the Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences (Shanghai, China). The HepG2, MHCC97H and MHCC97L cells were obtained from Chinese Academy of Medical Sciences (Beijing, China). HCCLM3, Huh7, HepG2, MHCC97H, MHCC97L were cultured in DMEM medium. BEL-7402, SK-Hep1, LO2 were cultured in RPMI 1640 medium. All the medium was added with 10 % fetal bovine serum (FBS) and 100 units/ml of penicillin and streptomycin (Hyclone, USA). All cells were grown in a humidified incubator with 5 % CO2 at 37 °C.
RNA preparation and quantitative real-time PCR
Total RNA from HCC tissue samples and adjacent non-tumorous tissue samples was extracted using Trizol Reagent (Invitrogen, USA) according to the manufacturer’s instructions. To determine mature miRNA expression levels, qRT-PCR was performed using a SYBR Premix Ex Taq (TaKaRa, Japan) on a Thermal Cycler Dice Real Time System (TaKaRa) with the following protocol: 30s at 95 °C followed by two-step PCR for 40 cycles of 95 °C for 5 s and 60 °C for 60s. MiRNA expression levels were normalized against the endogenous U6 small nuclear RNA (U6 snRNA) control. ADAM10 expression was measured by SYBR green qPCR assay and GAPDH was used as an endogenous control. The relative expression level of miR-655-3p in each paired tumor and adjacent non-tumorous tissue was calculated by the 2-ΔΔCT method. The sequences of the PCR primers were as follows: miR-655-3p forward, 5’-CCGCGATAATACATGGTTAACCTC-3’, and reverse primer was Uni-miR qPCR primer (TaKaRa); U6 forward, 5’-CTCGCTTCGGCAGCACA-3’ and U6 reverse, 5’-AACGCTTCACGAATTTGCGT-3’; ADAM10 forward, 5’-CTGCCCAGCATCTGACCCTAA-3’ and reverse, 5’-TTGCCATCAGAACTGGCACAC-3’; GAPDH forward, 5’CTCCTCCTGTTCGACAGTCAGC-3’, and reverse 5’-CCCAATACGACCAAATCCGTT-3’.
Oligonucleotides transfection
The miR-655-3p agomiR (agomiR-655-3p), antagomiR (anti-miR-655-3p), small interfering RNA for ADAM10 (siADAM10) and their negative control (Neg.Cont) Oligonucleotides used in this study were purchased from Shanghai GenePharma Co. Ltd. Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The sequences of Oligonucleotides were as follows: agomiR-655-3p, sense 5’-AUAAUACAUGGUUAACCUCUUU-3’ and antisense 5’-AGAGGUUAACCAUGUAUUAUUU-3’; miRNA negative controls, sense 5’-UUCUCCGAACGUGUCACGUTT-3’ and antisense 5’-ACGUGACACGUUCGGAGAATT-3’; anti-miR-655-3p, 5’-AAAGAGGUUAACCAUGUAUUAU-3’; negative control, 5’-UUGUACUACACAAAAGUACUG-3’; Si-ADAM10, 5’-CAGUGUGCAUUCAAGUCAA-3’.
Luciferase assay
The wild-type ADAM10-3’UTR(WT) and mutant ADAM10-3’UTR(MUT) containing the putative binding site of miR-655-3p were established and cloned in the Firefly luciferase expressing vector pMIR-REPORT (Obio Technology, China). Liver cancer cells were seeded into 24-well plates the day before transfection, and transfected with either the pMIR-REPORT-ADAM10-3’ UTR-WT or the pMIR-REPORT-ADAM10-3’ UTR-MUT reporter vector, together with the Renilla luciferase-expressing vector pRL-TK (Promega) and agomiR-655-3p or miR-Neg.Cont using Lipofectamine 2000 (Invitrogen). After 48 h, cells were harvested, and firefly and Renilla luciferase activities were measured using the dual-luciferase reporter assay system (Promega, Madison, WI).
Cell migration and invasion assays
After 48 h of transfection, cell concentration in each group was adjusted to 2 × 105 cells/mL with serum-free medium. The upper chamber of Transwell chamber (Costar; 24-well insert, pore size: 8 μm) was filled with 200 μl cell suspension, and the lower chamber was filled with 500 μL of medium supplementing 15 % FBS. For the invasion assay, polycarbonate filters coated with 50 μL Matrigel (1:9, BD Bioscience) were placed in a Transwell chamber. Three wells were used for each group. Cells were incubated for 24 h for the migration assay and 48 h for the invasion assay. Then, the cells on the upper surface were wiped slightly using cotton swabs, and the cells on the lower surface were fixed with 4 % paraformaldehyde and stained with 0.1 % crystal violet. The migratory cells were visualized and counted in five random visual fields per insert under an inverted microscope at 200× magnification (Nikon Microphot-FX, Japan).
MTT assay
After transfection, 5000 cells/well were seeded in 96-well plates in media containing 10 % FBS and incubated for 0, 24 h, 48 h, 72 h. On the indicated days, 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) (KyeGEN BioTECH, Nanjing, China) was added into each well according to the manufacturer’s instructions, and the cells were incubated for 4 h at 37 °C. The supernatants were then removed and 150uL of DMSO (Sigma-Aldrich, Germany) was added to per well to dissolve the formazan crystals. Absorbance levels were measured at the wavelength of 490 nm using an automatic microplate reader (Gene, HK). The data derived from triplicate samples are presented as mean ± s.d.
After transfection, 500 cells per well were counted and seeded in 6-well plates. The plates were incubated for 10 days, then the cells were fixed by 4 % paraformaldehyde and stained using 0.1 % crystal violet. Colonies were counted only if they included 50 cells at least. Triplicate independent experiments were performed and all the visible colonies were calculated manually.
Western blot
Cell samples were washed with ice-cold PBS and then lysed by RIPA (Beyotime, China) containing protease inhibitors (Beyotime, China). Cell protein lysates were separated in 10 % SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membranes were blocked by 5 % skim milk soluted in TBST buffers, and were incubated with primary antibodies for ADAM10, c-myc (Abcam, UK), E-cadherin, MMP9 (Santa Cruz Biotechnology, USA), β-catenin and cyclinD1 (ProteinTech Group, USA) overnight at 4 °C. PVDF membranes were washed in TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (ProteinTech Group, USA). Antibody against GAPDH (Cell Signaling Technology, USA) was used as an internal control. Antibody against Histone H3 (Abcam, UK) was used as an internal control for nuclear β-catenin. The protein of interest was visualized using ECL Western blotting substrate (Pierce, USA).
Immunohistochemical (IHC)
Formalin-stabilised liver tissue specimens were embedded in paraffin and cut into 4 μm sections for use in immunohistochemistry. After general deparaffinization, antigen retrieval was carried out for 30 sec with an autoclave using 0.01 mol/l sodium citrate buffer, pH 6.0. H2O2 (0.3 %) was used to block endogenous peroxidase activity for 30 min at 37 °C, and non-specific immunoglobulin binding sites were blocked by normal goat serum for 30 min at 37 °C. Sections were then incubated overnight with primary antibody(ADAM10,1:300, Abcam) at 4 °C, rinsed with PBS, and incubated with the appropriate secondary antibody for 30 min. The peroxidase reaction was developed with 3, 3-diaminobenzidine tetrahydrochloride (DAB).Sections were counterstained with Mayer’s hematoxylin, dehydrated, cleared in xylene, and mounted in Permount.
Statistical analysis
The statistical analyses were performed using the SPSS 17.0 software. Parametric data were presented as mean ± SEM, and differences between each group were analyzed using the Student’s t-test. The association between miR-655-3p relative expression and the clinicopathological parameters was evaluated by the χ2 test or Fisher’s exact test when appropriate. All of the p-values reported were two-sided, and significance was defined as p < 0.05.