Cell lines and culture
Human metastatic GC cell line BGC823 was gifted from Professor Q Guo, China Pharmaceutical University. The SGC7901 and MGC803 cell lines were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Human umbilical vein endothelial cells (HUVECs) were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). HUVECs, BGC823 and SGC7901 cells were cultured in RPMI-1640 (Hyclone, Thermo Scientific, Waltham, MA, USA), while MGC803 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) (Hyclone), 100 U/mL streptomycin and 100 μg/mL penicillin (Invitrogen, Carlsbad, CA, USA) in a humidified incubator at 37 °C with 5% CO2. Cycloheximide (CHX) (Sigma-Aldrich, St Louis, MO, USA) and MG132 (Selleck Chemicals, USA) were used at the indicated concentrations.
Plasmids, siRNAs and cell transfection
The wild-type Flag-SP1(WT) or its mutants [Flag-SP1 (K610R), Flag-SP1 (K624R), Flag-SP1 (K685R), Flag-SP1 (K693R) and Flag-SP1(ALL)] were inserted into the GV141 eukaryotic expression vector (Genechem, Shanghai, China) using XhoI/KpnI sites. Small interfering RNA used for TRIM25 gene knockdown was produced by Genomeditech (Shanghai, China). The sequences of siRNAs were as follows: si-TRIM25 1 (si-RNA 1), 5′-GAGUGAGAUCCAGACCUUGAA-3′ (forward) and 5′-UUCAAGGUCUGGAUCUCAACUC-3′ (reverse); si-TRIM25 2 (si-RNA 2), 5′-GAACUGAACCACAAGCUGAUA-3′ (forward) and 5′-UAUCAGCUUGUGGUUCAGUUC-3′ (reverse); si-TRIM25 3 (si-RNA 3), 5′-GUGCCCGAUUCCUCUUAGAGA-3′ (forward) and 5′-UCUCUAAGAGGAAUCGGGCAC-3′ (reverse). A scrambled siRNA was used as the negative control. Plasmids and siRNAs were transfected into cells using Lipofectamine 3000 (Invitrogen).
Mouse models
All animal experiments were in accordance with the Jiangsu Provincial Guidelines for the use of experimental animals, and it was approved by the Animal Care and Use Committee of Nanjing Medical University. Briefly, BALB/c nude mice (female, 6 weeks) were obtained from NLARSH China (Shanghai, China). Animals were housed in plastic cages and kept in a temperature-, humidity-maintained, and light-controlled room (23 ± 1 °C; 50 ± 5% humidity; 12 h light/dark cycle starting at 7:00 am). They had ad libitum accesses to food and water.
For establishing xenograft mouse models, 5 × 106/200 μL of BGC823 cells were subcutaneously injected into flank of BALB/c nude mice. When the tumor volume (V = 0.5 × length × width2) reached about 100 mm3, the mice were randomly divided into different groups and received the indicated treatments. General situations and activities of each mouse were daily recorded. Tumor sizes were recorded by caliper every 3 days for calculating tumor volumes. Body weight of the experimental mouse was also recorded every 3 days. The endpoint of the experimental therapy was determined until enough growth (up to 2000 mm3) of the xenograft tumor or intolerable side effects.
In the present study, the strategy to screen JWA protein based functional tumor inhibitory peptides included four steps, (1) all the candidate fragments were selected from non-transmembrane regions and contain amino acid can be phosphorylated in its sequence; (2) the intra-tumor injection in xenograft GC tumor in mouse model was used for first round screening for all candidate fragments; (3) the identified functional peptides in (2) were modified with HWGF as MMP2 target and used for secondary screening by intraperitoneal injection in xenograft GC mouse model; (4) the functional peptides selected in (3) were further used to inhibit metastasis of GC (Fig. 1a).
For establishment of metastatic GC mouse model, five-week-old BALB/c nude mice were randomly divided into two groups, 3 mice each. BGC823 cells were suspended in PBS. The mice were injected intravenously with 2 × 106 cells in 0.15 mL of PBS through tail vein. On next day, the mice were treated by intra-peritoneal (ip) injection of Ctrl-H-P and JP3 and followed by luciferase substrate (GM-040611, Genomeditech, China); and photographed using the IVIS Spectrum in vivo imaging system (PerkinElmer, USA) on day 50th to observe the cancer cell metastasis.
Designing and synthesis of polypeptides
According to the predicted phosphorylation sites in 188 amino acids of JWA, six fragments (Fig. 1b) were selected and finally ten peptides were synthesized since only a single phosphorus modification to one amino acid residue was designed for each peptide. Both PJP3 and PJP6 fragments were split in three modified ones from each since three modifiable amino acid residues were contained in its sequences, respectively. A routine acetylation modification to each peptide was conducted to generate N-acetylation and C-amide, thereby increase its in vivo stability. According to the full length of JWA amino acid sequence and spatial structure, each synthesized peptide with a phosphorylated serine (S), threonine (T) or tyrosine (Y) in the center, and extension of 3–7 amino acid sequences in both sides. The MMP2-targeting motif HWGF was added after a four glycine (G) linker at C- termini of peptide (named as JP1-JP3).
All the peptides were synthesized by GL Biochem Ltd. (Shanghai, China), with a purity of more than 98%. These solid-phase synthesized peptides have good water solubility. The synthesized peptides were packed as 10 mg in sterile Eppendorf tube and always kept at − 20 °C. All peptides were dissolved in pre-cold PBS to the indicated doses/concentrations before use.
Immunofluorescence assay
GC cells were treated with different concentrations of FITC-JP3 for different time points, followed by washing with phosphate buffer saline (PBS) for 3 times. Cells were then fixed with methanol for 15 min at room temperature and washed in PBST (PBS supplemented with 0.5% Tween-20). Cell nuclei were counterstained with DAPI (Beyotime, Jiangsu, China) for 20 min. The confocal images of stained cells were captured using the Zeiss AIM software on a Zeiss LSM 700 confocal microscope system (Carl Zeiss Jena, Oberkochen, Germany).
Coimmunoprecipitation (co-IP)
BGC823 and SGC7901 GC cells were grown to confluence and processed for coimmunoprecipitation by standard procedures as previously described [16]. The pre-cleared lysate was incubated with anti-SP1 or anti-TRIM25 polyclonal antibodies for 1 h, and then incubated with protein A/G agarose beads overnight. Collected beads by centrifugation were washed for 5 times with washing buffer, and resuspended in 1× sodium lauryl sulfate loading buffer. Immunoprecipitation was eluted by incubation at 95 °C for 5 min.The eluted proteins were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis, and then Western blotting with anti-SP1 (CST, Danvers MA, USA), anti-TRIM25 and anti-ubiquitin antibodies, respectively.
Ubiquitination assay
GC cells were treated with or without JP3, followed by incubation with MG132 (10 μM) for another 6 h. Cells were harvested, prepared for protein samples and divided for Western blotting and co-IP assay, respectively. For co-IP assay, the protein (500 μg) was cultured with anti-SP1 or anti-TRIM25 antibody (dilution: 1:250) at 4 °C for 1 h, and Protein A/G Plus-Agarose overnight. After three cycles of washing in pre-cold IP buffer and centrifugation at 4 °C, 1000×g for 5 min, the immunoprecipitate was collected by centrifugation, eluted and examined by Western blotting.
Western blotting analysis
Western blotting analysis was carried out as previously described. The antibodies against MMP2, Tubulin, Ub, HA-tag, Flag-tag and Ki-67 were purchased from Cell Signal Technology (Danvers MA, USA); anti-CD31 and PCNA were provided by Epitomics (MA, USA); anti-GAPDH and Actin were purchased from Beyotime (Jiangsu, China); anti-TRIM25 and SP1 were provided by Proteintech (Wuhan, China); anti-His was purchased from MBL (Japan). All above antibodies were used at a dilution of 1:1000.
Quantitative real-time polymerase chain reaction (qRT-PCR)
According to the manufacturer’s protocol, total RNAs were isolated using TRIzol (Invitrogen, CA, USA) and reversely transcribed into cDNAs using reverse transcription kit and the SYBR Green Master Mix kit (Takara, Otsu, Japan). The complementary DNA (cDNA) was amplified with the following primers: 5′-GTCTCTACCCAGAACAGTTTCC-3′ (forward) and 5′-ATCCAACACAGGCTGATTCC-3′ (reverse) for TRIM25; 5′-GCCGGTGCTGAGTATGTC-3′ (forward) and 5′-CTTCTGGGTGGCAGTGAT-3′ (reverse) for GAPDH; 5′-GGTGCCTTTTCACAGGCTC’-3′ (forward) and 5′-GCTGTTCTCATTGGGTGACTC-3′ (reverse) for SP1. Quantitative RT-PCR was carried out an ABI Prism 7900 Sequence detection system (Applied Biosystems, Canada). Relative levels were normalized to that of GAPDH.
HUVECs tube formation assay
After 6 hours of peptide treatment of GC cells, the medium was replaced and cultured in a 60 mm petri dish containing 1% serum for 24 h. On the next day, 2 mL of conditional medium was collected. For tube formation assay, a 96-well plate was coated with 50 μL of Matrigel™ (BD Biosciences) and kept at 37 °C for 2 h. Then, 1.2 × 104 HUVECs suspended in 100 μL of conditional medium were applied to each well of the precoated 96-well plate. After incubation at 37 °C for 4 h, tubular structures formed in the Matrigel were captured in five randomly selected fields per sample under a microscope.
Protein docking analysis
I-TASSER (Iterative Threading Assembly Refinement) was designed for protein structure modelling by iterative threading assembly simulations. The molecular structures of JP3, MMP2, and TRIM25 were constructed by I-TASSER. The largest possible binding pocket of MMP2 and TRIM25 was then predicted by Discovery Studio 3.0. These predicted pockets were utilized to construct an initial coarse model of the JP3-MMP2, JP3-MEK1/2 and JP3-TRIM25 complex. The coarse model of a complex was then refined using RosettaDock. Since a peptide was considered as a rigid body in RosettaDock, the peptide–protein complex with the lowest energy was then refined by utilizing the FelxPepDock module of Rosetta. The output of 2000 models was then ranked based on their energy scores. The model with the lowest energy was chosen for further research, i.e., binding sites and electrostatic properties. The binding sites between proteins were obtained based on space coordinates and types of amino acid (hydrogen bond: ≤3.5 Å; ionic: ≤3.5 Å; disulphide: ≤2.5 Å; van-der-waals: ≤0.5 Å; pi-pi interaction: ≤6.5 Å). Electrostatic interactions were found in almost all biomolecular systems and processes. To predict potential phosphorylation sites of TRIM25, a visual inspection of the electrostatic potential at a solvent-accessible surface was used. The electrostatic properties of the structures were calculated with the Adaptive Poisson–Boltzmann Solver (APBS) and PDB2PQR. High-quality 3D images of proteins were drawn with PyMOL/VMD.
TMA construction and assessment of IHC
The construction of the GC TMAs was performed with standard procedures, and the assessment of the IHC employed a semiquantitative immunoreactivity score (IRS) as reported elsewhere [17]. The concordance for the IRS of the TRIM25 and SP1 staining scores between the two pathologists was 81 (90%) in 90 of the TMA cohort. The optimum value of cutoff points of the TRIM25 or SP1 IRS in this study were both 6 because the predictive value of this cutoff point for death was the best in the GC cohorts. Under these conditions, the samples with IRS scores 0–6 and IRS 8–12 were classified as low and high expressions of TRIM25 and SP1, respectively.
Statistical analysis
All analyses and depicted graphs were performed with GraphPad Prism 6.0 Software (GraphPad Inc., San Diego, CA, USA). Data were expressed as mean ± SEM. The Student’s t-test was used to analyze differences between groups, while one-way ANOVA was used when more than two groups. In all statistical comparisons, *P value < 0.05 were considered as statistically significant (*P value < 0.05, **P < 0.01 and ***P < 0.001).