Study population and specimens
A total of 245 samples were obtained from surgically treated HCC patients who had not receive preoperative treatments at West China Hospital (Chengdu, China) from 2009 to 2013. Of them, 108 samples contained HCC and paired adjacent tissues, were used for investigating the expression difference between tumor and adjacent tissues. The total 245 samples were used for quantification of NCSTN expression and analysis of their correlation with patient outcomes after hepatectomy. This study has been approved by the ethics committee of West China Hospital.
Tissue microarray and immunohistochemistry (IHC)
The tissue microarrays and IHC analysis were performed as we previously described(11). Two independent pathologists evaluated the staining. Quantitative analyses were performed by summing the staining intensity score (0, negative; 1, weak; 2, moderate; 3, strong) of ten randomly selected fields. The cut-off value for classification of patients was 150 points. The antibodies for IHC were shown in Supplementary table 1.
Western blotting (WB)
Tissues and cells were lysed in RIPA buffer, which was supplemented with protease and phosphatase inhibitor (Thermo Fisher Scientific, CA, USA), and then quantified by using BCA protein assay kit (Beyotime Biotechnology, China). The WB assay was conducted as we previously described(12). The intensity of signals was scanned using ChemiDoc MP Imager System (Bio-Rad, California, USA). The primary antibodies for WB were shown in Supplementary table 1.
The Cancer Genome Atlas (TCGA) data analysis
High-throughput RNA-sequencing data of 370 HCC samples and 50 matched adjacent samples were downloaded from the TCGA dataset. The software of X-tile (version 3.6.0, Yale University School of Medicine) was used for identification of optimal cut-off value for NCSTN based on the association between mRNA expression level and overall survival (OS).
Cell culture and reagents
Human HCC cell lines Hep3B, Huh7, HepG2 and HCCLM3 were purchased from Cell Bank of Shanghai Institutes for Biological Science, Chinese Academy of Science (Shanghai, China), SNU449 and SNU387 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultivated in recommended medium, supplemented with 10% FBS, 100 µg/mL streptomycin and 100 U/mL penicillin, at 37 °C with 5% CO2. The Notch inhibitor FLI-06 and AKT inhibitor MK-2206 2HCl were obtained from Selleck (Houston, TX, USA).
Lentiviral vector containing human NCSTN sequence and lentiviral particles containing shNCSTN-1 and shNCSTN-2 were synthesized by GenePharma (Shanghai, China). The specific small interfering RNA (siRNA) oligonucleotides targeting β-catenin and Zeb1 were synthesised by Viewsolid Biotech (Beijing, China). 1 × 106 cells were seeded in 6-well plate and transfected with specific siRNA using GenmuteTM Reagent (SignaGen Laboratories, Maryland, USA) according to the manufacturer’s instructions, and then harvested 48 hours post transfection.
In vitro cell proliferation and cell cycle assays
2 × 103 cells within 100 µl medium were seeded in triplicate wells of 96-well plate and observed for 120 hours. Each well was co-cultured with 10 µl Cell Counting Kit-8 (CCK-8) (Beyotime Biotechnology, Shanghai, China) and incubated at 37 ℃ for 2 hours. The absorbance was detected at 450 nm by using the EonTM Microplate Reader (BioTek, VT, USA). 1 × 103 cells were seeded in triplicate wells of 6-well plate and cultured for 2 weeks. After fixed with 4% paraformaldehyde for 20 min, the colonies were stained using 0.1% crystal violet and counted. According to the manufacturer’s instructions of Cell Cycle Kit (4A Biotech, Beijing, China), cells were fixed using 95% cold ethanol, followed by incubation with propidium iodide at 37 °C for 30 min and analyzed by the CytoFLEX Research Flow Cytometer (Beckman Coulter, CA, USA).
In vitro wound-healing assay
Cells were plated in triplicate into 6-well plates. A standard 10 µl pipette tip was used to scratch wound when the cells reached a density of 95%. Subsequently, the cells were cultured in FBS-free medium. After 24 or 48 hours, the wound closure was captured by a microscope and calculated using the software of Image J (National Institutes of Health, Bethesda, MD, USA).
In vitro transwell migration and matrigel invasion assay
Transwell chambers (8.0 µm pore size, Corning Costar, Kennebunk, USA) were applied in migration and matrigel invasion assays. For migration assay, 3 × 104 cells were suspended in FBS-free DMED medium and then seeded into upper chamber. For matrigel invasion assay, 5 × 104 cells were suspended in FBS-free medium and seeded into upper chamber with matrigel coating. DMEM medium containing 10% FBS were added to the lower chamber. After cultivation for 24 or 48 hours, cells which migrated or invaded onto the lower surface of lower chambers were fixed using 4% paraformaldehyde, followed by stained using 0.1% crystal violet. Cells of 5 randomly selected fields were calculated and counted using Image J.
In vivo xenograft assay
The in vivo animal experiment was authorized by the Animal Ethic Review Committees of the West China Hospital. 5-week old male BALB/c nude mice were obtained from HFK BIOSCIENCE (Beijing, China) and used for in vivo assays. 1 × 106 cells (HCCLM3) or 2 × 106 cells (Hep3B) were suspended using 100 µl PBS and subsequently implanted subcutaneously into right axillas of mice (5 mice for each group). Measurement of tumor size was performed weekly by using the formula of length × width2 × 0.52. The mice were sacrificed at 4 or 6 weeks after implantation, followed by measure of tumor weight. For liver orthotopic-implanted HCC models, 1 × 106 cells were implanted into left lobe after mice were anesthetized. Mice were sacrificed 4 weeks after implantation and the livers were scanned using the [email protected] Lumina II system (Caliper Life Sciences, Hopkinton, MA, USA). 2 × 106 cells were used for construction of lung metastasis models by injection into tail veins and the mice were sacrificed at the time of 6 weeks post implantation and the lungs were scanned using the [email protected] Lumina II system. The metastatic foci were confirmed by hematoxylin and eosin staining.
RNA extraction and quantitative real-time PCR
Total RNAs of HCC cells were extracted by using Cell Total RNA Isolation Kit (Foregene, Chengdu, China) in accordance with the manufacturer’s instructions. The first-strand complementary DNA was synthesized using HiScript II Reverse Transcriptase (Vazyme, Nanjing, China). ChamQ™ [email protected] qPCR Master Mix (Vazyme Biotech, Nanjing, China) was utilized for real-time PCR. All the reactions were performed in triplicate and the relative mRNA expression levels were quantitated using the 2−ΔΔCT method. U6 was utilized as endogenous control. Primers used in this study were summarized in Supplementary table 2.
Immunofluorescence (IF) analysis
3 × 103 cells were plated on cover slips in 24-well plates. The cells were fixed using 4% paraformaldehyde at room temperature for 20 minutes, premeabilized using 0.2% Triton X-100 in PBS for 10 minutes and then blocked with 5% BSA at room temperature for 1 hour, followed by incubation of primary antibodies at 4 ℃ overnight. Then the cells were washed using PBST (0.1% Tween-20 in PBS) for tree times and incubated with appropriate fluorophore-conjugated secondary antibodies (1:1000, Thermo Fisher Scientific, CA, USA). After incubation with DAPI (Kaiji, Nanjing, China), the cover slips were mounted on slides and scanned using AX10 imager A2 microscope (Carl Zeiss MicroImaging).
Subcellular protein fractionation
Cytoplasmic and nuclear protein fractions were extracted by using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, CA, USA) according to its protocol. The extracted protein samples were subsequently analyzed by WB assay. The β-Tubulin and Histone H3 were used as cytoplasmic and nuclear endogenous control, respectively.
TOP/FOP Flash assay
1 × 104 cells were seeded in triplicate in a 24-well plate 24 hours prior to transfection. They were co-transfected with the pRL-CMV plasmid (Renilla luciferase, Promega) plus either TOP-Flash or FOP-Flash plasmid (Upstate) according to the instructions of Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). Cells were cultured for 48 hours and then the luciferase activity were evaluated using Duo-Luciferase HS Assay Kit (GeneCopoeia, CA, USA) in accordance with the manufacturer’s instructions. The results were shown as TOP/FOP Flash activity.
Co-IP was conducted using Pierce Crosslink Magnetic IP/Co-IP Kit in accordance with the manufacturer’s instructions (Thermo Fisher Scientific, CA, USA). Briefly, cell lysates were incubated with protein A/G magnetic beads which were previously bind to primary antibodies, then dissociated the bound antigens from antibody-crosslinked beads by eluting in a low-pH elution buffer. The eluent was detected by WB analysis.
All the statistical analyses were performed by applying SPSS software (version 23.0, SPSS Inc., Chicago, IL, USA), GraphPad Prism software (version, 8.0La Jolla, CA, USA) and MedCalc software (version 15.2.2). Data was exhibited as mean ± standard deviation (SD). Student’s t-test was used to investigate continuous variables. Multiple hypothesis test was assessed by using Monte Carlo method. The Person χ2 test was applied to examine correlations. The survival curves were plotted using Kaplan-Meier method and tested by log-rank test. Subsequently, Cox proportional hazards regression model (enter method) was utilized to evaluation of potential independent prognostic factors. Potential confounders with P values less than 0.05 in univariate regression analyses were selected for multivariate regression models. A two-tailed P value < 0.05 was considered statistically significant.