Tissue specimens and Immunohistochemistry
A total of 66 paired specimens of tumor and adjacent non-tumor tissues were collected from 66 HCC patients who underwent hepatectomy at the Hepatic Surgery Center, Tongji Hospital of Huazhong University of Science and Technology (HUST) (Wuhan, China). Matched fresh specimens of HCC tissues and adjacent non-tumorous liver tissue were lysed separately for western blot analysis. A tissue microarray of 110 pairs of primary HCC tissues with their clinical and prognosis data were acquired from the specimen library of the Hepatic Surgery Center, Tongji Hospital of Huazhong University of Science and Technology. The samples were obtained in surgeries from 2012/2/16 to 2014/4/1, and the database has been updated every month since 2012/2/16. All HCC tissues were identified in Tongji hospital's department of pathology. Before surgery, there was no local or systemic therapy. After surgery, no anticancer treatment was conducted before recurrence. Written informed consent was obtained from all patients and each procedure was approved by the Ethical Committee of Tongji Hospital. The fundamental processes of the Immunohistochemistry assay have been mentioned previously. The image scores were evaluated by 3 different pathologists ignorant of patient clinical pathological characteristics. The total score of each image was calculated by multiplying the score of staining area percentage by intensity score as mentioned previously. The total scores of ≥ 5 was defined as positive; Otherwise, defined as negative.
Cell lines and cell culture
The lentivirus packaging cell line 293T, human fetal liver cell line HL-7702, hepatoma cell line HepG2, HCC cell lines Hep3B, Huh7, SK-Hep1, HLF, HLE, HCCLM3, PLC/PRF/5, 97H and Bel7402 were purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China). The cell lines were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, ThermoFisher Scientific, Waltham, Massachusetts, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, North America) at 37°C in 5% CO2 and 95% air.
Reagents and Antibodies
Rat tail Collagen I (BD Bioscience, MA, USA), DDR1 inhibitor imatinib and ponatinib (MedChemExpress, NJ, USA). Puromycin, trypsin-EDTA, Opti-MEM medium and polybrene were obtained as previous described. Lipofectamine 2000 Reagent and Lipofectamine 3000 Reagent were purchased from Invitrogen (Life Technologies, Carlsbad, CA, USA). CHX CQ and Mg132 were obtained from MedChemExpress (Shanghai, China). NH4Cl was obtained from promoter company (promoter, Wuhan, China). All antibodies used in the project were detailed in Supporting Table S1.
Plasmid construction and small interference RNAs
The human DDR1 (NM_001954.4) cDNA, and pcDNA3.1 plasmid were gifts from the Hepatic Surgery Center, Tongji Hospital, Huazhong University of Science and Technology, P .R. China. pBABE-puro (Plasmid #1764), gag/pol (Plasmid #14887), pMD2.G(Plasmid #12259), pLKO.1-TRC cloning vector (Plasmid # 10878), psPAX2(Plasmid #12260) were purchased from Addgene (Addgene, Cambridge, MA, USA). To establish pBABE-Flag-DDR1 or pBABE-Flag-SLC1A5 plasmid, the human cDNA was cloned into the BamHI/EcoRI site of the pBABE-puro retroviral vector, and was identified by sequencing (TSINGKE, Wuhan, China). To construct pLKO.1-scramble, pLKO.1-shDDR1, and pLKO.1-shSLC1A5, the target double-stranded oligonucleotides (shRNA) sequences and one non-targeting sequence (negative control, scramble) were annealed and cloned into the AgeI/EcoRI site of pLKO.1 vector. The sequences of target shRNA oligo pairs are listed in Supporting Table S2. Viral production, infection, establishment of stable cell clones were described previously. pcDNA3.1 plasmid inserted by Flag-, Myc- or HA- tagged DDR1 and its mutants, Flag- or HA-tagged SLC1A5, Flag-tagged PPP2R1A, SRFBP1 and CUL4B were constructed according to ClonExpress II One Step Cloning Kit and Mut Express II Fast Mutagenesis Kit V2 (Vazyme, Nanjing, China) protocol and were identified by sequencing (TSINGKE, Wuhan, China).
Cell growth assay
Sk-hep1-DDR1 (1.5*103cells/ well), HepG2-DDR1 (1.5*103 cells/ well), HLF-shDDR1 (1*103cells/well), HLE-shDDR1 (1*103cells/well), Sk-hep1-SLC1A5 (1.5*103cells/ well), HepG2-SLC1A5(1.5*103cells/well), Hep3B-shSLC1A5(2*103cells/well), PLC/PRF/5-shSLC1A5(2*103 cells/ well) and the same amount of their control cells were cultured in 96-well plate. At the indicated time points, the Cell Counting Kit-8 (CCK-8, Beyotime Institute of Biotechnology) was added to the cells for 2 hours, and then the plate was read using an enzyme-linked immunosorbent assay plate reader (Bio-Tek Elx 800, USA) at 450 nm. Cells (500 cells/well) were plated in each well of a 6-well plate. At day 14 the plates were fixed in 4% paraformaldehyde, stained with 1% crystal violet, and the numbers of colonies greater than 100µm in diameter were counted and analyzed by Image J 1.52a(NIH, USA).
Cell cycle analysis
For cell cycle analysis, the cells were plated in a 6-well culture plate and grown for 24 hours. Then incubate the cells with 1 mM thymidine(Sigma-Aldrich) for 24 hours to synchronize the cells at the G1/S or G2/M boundary. The cells were then treated with fresh medium containing 2.5 lg/mL TM for different lengths of time. Next, the cells were trypsinized, washed twice with cold PBS, and fixed with cold 70% ethanol overnight at 20°C. The cells were then washed twice with PBS and incubated with a PBS solution of 10 mg/mL RNase A, 400 mg/mL propidium iodide, and 0.1% Triton X at room temperature (RT) for 30 minutes. The cells were then analyzed by flow cytometry.
Immunoprecipitation and western blotting
Immunoblotting assay and co-immunoprecipitation assay were performed as described previously.Briefly, cells were collected and lysed on ice with IP lysis buffer. lysates were incubated with protein G agarose for 2 hours, and immunoprecipitated with indicated antibodies at 4°C overnight. Then lysates were incubated with protein G agarose for 1 hours followed by 1wash using IP lysis buffer and 3 washes using washing buffer (300 mM NaCl, 1.0 mM EDTA, 25 mM Tris-HCl, pH7.4, 1.0% NP-40). The beads were eluted with 2×SDS-PAGE loading buffer and then subjected to immunoblotting analysis.
Reverse transcription PCR and Real-time quantitative PCR
Total cell RNA was extracted with TRIzol Reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA). Reverse transcription was carried out with the QuantScript RT Kit (TIANGEN, Beijing, China) according to manufacturers’ introductions. Real-time fluorescence quantitative PCR was performed with the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using SuperReal PreMix Plus (SYBR Green) kit (TIANGEN, Beijing, China) according to the manufacturer’s protocol (Each gene expression level was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of the same sample. Each sample was done in triplicate independently. The primers are listed in Supporting Table S3.
293T cells transiently transfected with FLAG-DDR1 or FLAG-vector were lysed in IP lysis buffer (25mM Tris-HCl (pH 7.4), 150mM NaCl, 1% NP-40, 1mM EDTA, 10% Glycerol and protease inhibitor cocktails) and IP assays were performed as described previously. The eluted proteins were separated by SDS-PAGE and then sent to mass spectrometry for detection. Mass spectrometry was detected and analyzed by ptm-bio lab (PTM BIO, Hangzhou, China).
Immunofluorescence assay was performed as described previously. In brief, after the indicated treatments, cells were cultured on coverslips for 12h, fixed in 4% paraformaldehyde for 15 min at room temperature, and permeabilized with 0.5% Triton X-100 for 20 min. After blocking, the slides were incubated with according primary antibody overnight at 4°C in a humidified box. After that, the slides were then washed three times and incubated with according secondary antibody for 4 h at room temperature in a humidified box. Finally, cell nuclei were stained by 40, 60-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) for 5 min. Pictures were obtained by phase-contrast and confocal laser-scanning microscopy.
Subcutaneous xenograft study in nude mice
Four-week-old male BALB/c (nu/nu) mice were raised under specific pathogen-free conditions. All animals were cared for according to the Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by the Committee on the Ethics of Animal Experiments of the Tongji Medical College, HUST. Mice were subcutaneously injected with 4 × 106cells and tumor size was measured every 4 days. The volume of tumor was calculated by the formula: volume = 0.52× (long diameter) × (short diameter)/2. Tumors were excised at 30 days post injection followed by photograph and weigh. After that, tumors were fixed with 4% paraformaldehyde and applied to immunohistochemical analysis.
Data analysis was performed using Prism 6.0 (GraphPad Software, La Jolla, CA, USA) software. The results were presented as the mean ± SEM. The difference between two groups were analyzed by two-tailed Student's t-test, ANOVA test, or a nonparametric test. χ2 test or Fisher’s exact test was used to analyze categorical data. Association between DDR1 and SLC1A5 expression in HCC tissues was calculated using Pearson correlation test. The survival between subgroups was assessed by Kaplan-Meier and log-rank analysis. Three independent experiments were carried out to insure repeatability. A value of P < 0.05 was regarded as statistically significance.