Cell lines and cell culture
Human CRC (HCT)-116 cell line (Cell Bank, Chinese Academy of Sciences, Shanghai, China) was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 ug/ml). EpCAM+CD44+HCT-116 cells (CCSCs) were cultured in Dulbecco's modified Eagle's medium with Ham’s F12 (DMEM/F12) containing 2% B27, 20 ng/ml basic fibroblast growth factor (bFGF), and 20 ng/ml epidermal growth factor (EGF) as we previously described . The cells were cultured in an incubator at 37°C containing 5% CO2.
A total of 35 6-week-old female athymic BALB/c nude mice (20±2 g) were purchased from Beijing HFK Bioscience Co., Ltd. and housed in the Laboratory Animal Center at Jilin University. All animals bred in specific pathogen-free conditions, with 25±2℃ temperature, 55±5% humidity and a 12-h light/ dark cycle as the same conditions previously described . Animals had ad libitum access to water and mouse chow diet. An acclimation period of at least 1 week was implemented for all mice prior to use in experiments. All the procedures were approved by Animal Care Committee of Jilin University (No. 2019-0046) and performed according to the Jilin University Guidelines for Animal Research.
Reagents and antibodies
The RPMI 1640 and DMEM/F12 were bought from Gibco (Grand Island, NY, USA) and the bFGF and EGF were purchased from PEPROTECH (Rocky Hill, NJ, USA). Anti-human CD44-biotin and anti-human epithelial cell adhesion molecule (EpCAM) for magnetic-activated cell sorting (MACS), were both purchased from eBioscience (San Diego, CA, USA). Phycoerythrin (PE)-anti-human CD44 and fluorescein isothiocyanate (FITC)-anti-EpCAM (eBioscience) were used for flow cytometric analysis. Mouse anti-Nanog and anti-GAPDH antibodies (Biolegend, San Diego, CA, USA), mouse anti-MMP-9, mouse anti-MMP-2, mouse anti-Bax, mouse anti-Bcl-2, rabbit anti-cleaved caspase-3, rabbit anti-TIMP-1 were purchased from Abcam (Cambrige, UK) using for western blotting.
Magnetic activated cell sorting (MACS)
As we previously described, the experiment of MACS was performed using a CELLection™ Biotin Binder kit following the manufacturer's instructions .In brief, HCT-116 cells were collected and incubated with anti-human EpCAM-biotin at 4°C for 10 min. Subsequently, the cells were labeled with dynabeads (500 µl) for 20 min at 4°C, and a magnet was used to obtain the labeled cells (EpCAM+ cells). EpCAM+ cells were then incubated in releasing buffer (Dnase I; 16 µl) for 15 min at room temperature (RT) and collected. EpCAM+ cells were incubated with anti-human CD44-biotin for 10 min, and subsequently with dynabeads (50 µl) for 20 min. Target cells (EPCAM+CD44+ CCSCs) were separated using a magnet after adding releasing buffer (Dnase I; 4 µl) into the cells and incubating for 15 min at RT. EpCAM+/CD44+ CCSCs were freshly prepared for use.
Silencing by siRNA transfection
To inhibit Nanog expression in the EpCAM+ CD44+ HCT-116 cells (CCSCs), silencing by small interfering RNAs (siRNAs) was performed using riboFECTTM CP Transfection Kit (Ribobio, Guangzhou, China) according to the manufacturer’s instructions. Nanog siRNA and its negative control siRNA were designed and synthesized by Riobobio (Guangzhou, China). Nanog siRNA sequence is 5’-AACTATCCATCCTTGCAAA-3’. The CCSCs were first cultured in 6-well plates (106 cells/well) in DMEM/F12 medium for 48 h, and then transfected with 20 µM Nanog siRNA or negative control siRNA. Cells that had not been transfected served as controls. After transfection for 48 h, the cells were cultured for further evaluation.
The cells (106/tube) were washed twice with phosphate-buffered saline (PBS) and incubated with PE-anti-human CD44 and FITC-anti-human EpCAM (appropriate dilution per antibody) at 4°C for 20 min. Subsequently, the cells were washed again with PBS twice. The labeled cells were analyzed using a flow cytometer (BD Biosciences).
To induce differentiation, CCSCs were cultured in DMEM/F12 medium supplemented with 10% FBS for 3 days. The results were observed using an inverted microscope.
Single-cell colony formation
EpCAM+ CD44+HCT-116 cells were seeded in DMEM/F12 medium at a density of 200 cells per well on 6-well plates and cultured at 37°C for 3 weeks. The medium was replaced every 2 to 3 days. Plates were photographed with an optical inverted microscope.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total cellular RNA was extracted using TRIzol reagent (Invitrogen, USA); reverse transcription was performed from 1 µg of the total RNA using PrimeScript RT Master Mix (TaKaRa, Dalian, China), according to the manufacturer's instructions. qRT-PCR was performed with TransStart Top Green qPCR SuperMix (TransGen, Beijing, China) using a real-time PCR system (PikoReal 96, ThermoFisher Scientific, USA) with the following program: 94°C for 30 sec, followed by 40 cycles of amplification (94°C for 5 sec, 60°C for 15 sec, and 72°C for 1 sec). The primer sequences (GeneCreate, Wuhan, China) used for quantitative real-time PCR are shown in Table 1. GAPDH was used as an endogenous control. The relative expression levels of mRNA transcripts were analyzed by the 2−ΔΔCt method. All the experiments were performed in triplicate.
Western blot analysis
The CCSCs transfected with Nanog siRNA or negative control siRNA were harvested 48 h after transfection. For western blot analysis, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor (DINGGUO, Beijing, China). After quantification of protein concentrations using a BCA protein assay kit (Beyotime, Nanjing, Jiangsu, China), equivalent quantities of protein (30 μg/well) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated in blocking solution (5% non-fat milk/TBST), and then incubated with the following primary antibodies overnight at 4°C: mouse anti-Nanog (1:1000), mouse anti-MMP-9 (1:500), mouse anti-MMP-2 (1:500), mouse anti-Bax (1:500), mouse anti-Bcl-2 (1:500), rabbit anti-cleaved caspase-3 (1:500), rabbit anti-TIMP-1 (1:500) and mouse anti-GAPDH (1:2000). After being washed three times with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at RT. Then, the membranes were washed again three times with TBST. The protein bands were detected using enhanced chemiluminescence detection kit (ECL; Beyotime, Nanjing, Jiangsu, China) on ChemiDoc XPS system (Bio-Rad, USA), and GAPDH was used as the loading control. Protein expression was quantified using Image Lab 5.2.1 (Bio-Rad) and normalized to GAPDH.
MTS cell proliferation assay
Cell proliferation was assessed using CellTiter 96 Aqueous assay kit (Promega; Madison, WI, USA). The CCSCs transfected with Nanog siRNA or negative control siRNA were plated at a density of 104 cells/well into 96-well plates (100 µl medium/well). After 48 h, 40 µl of 3-(4,5-dimethylthiazol-2-yl)-5-(3-C-arboxymethoxyphenyl)-2-(sulfophenyl)-2H-tetrazolium solution (MTS) was added to each well according to the manufacturer's instructions, followed by incubation of the plates at 37°C, 5% CO2 for 2 h, and the absorbance was measured at 490 nm using a microplate reader. All experiments were performed in triplicate and repeated three times.
Annexin V analysis
Annexin V analysis was performed using the -Annexin V-FITC kit (KeyGEN, Nanjing, Jiangsu, China) according to the manufacturer's instructions. Briefly, after CCSCs were transfected with Nanog siRNA or negative control siRNA for 48 h, they were harvested and washed twice with PBS and then re-suspended in binding buffer at a density of 1×106 cells/ml. Subsequently, Annexin V-FITC (5µl) and propidium iodide (PI) (5µl) were added to the cells (500 µl). After being incubated for 15 min in the dark at RT, the cells were analyzed using a flow cytometer (BD Biosciences, USA). Annexin V-/PI- cells present cell survival, Annexin V+/PI- cells were shown cells in early apoptosis, and Annexin V+/PI+ cells were in late apoptosis or necrotic. The experiments were repeated independently three times.
Alterations in mitochondrial membrane potential were measured by flow cytometry using the JC-1 kit (KeyGEN, Nanjing, Jiangsu, China) according to the manufacturer's instructions. Briefly, after CCSCs were transfected with Nanog siRNA or negative control siRNA for 48 h, the cells were harvested and washed twice with PBS and re-suspended in 500 µl incubation buffer containing the JC-1 dye (1 µl) at a density of 1×106 cells/ml. After being incubated for 15 min at 37°C, 5% CO2, the cells were collected and washed twice with the incubation buffer. Subsequently, cells were re-suspended in 500 µl incubation buffer and analyzed using a flow cytometer (BD Biosciences, USA). Annexin V-/PI- cells present cell survival, Annexin V+/PI- cells were shown cells in early apoptosis, and Annexin V+/PI+ cells were in late apoptosis or necrotic. The experiments were repeated independently three times.
Transwell invasion assay
The invasion assays were performed using 6.5-mm diameter Transwell plates (8 µm pore size, Corning, Steuben County, NY, USA) coated with a thin layer of Matrigel (BD Biosciences, San Diego, CA, USA), through which invading cells could migrate and eventually attach to the bottom of the polycarbonate layer. The CCSCs were resuspended in serum free DMEM/F12 at a concentration of 1×106/ml. The upper chamber was loaded with 100 µl of cell suspension and the lower chamber was loaded with 500 µl of DMEM/F12 medium supplemented with 10% FBS as the chemoattractant. After incubation for 24 h at 37°C and 5% CO2, non-invading cells in the upper chamber were removed with a PBS-soaked cotton swab and the cells that had invaded the membranes were stained with 0.5% crystal violet and counted under a light microscope. Each assay was replicated 3 times. The invaded cells were counted under the microscope in five random fields in each chamber. The assay was performed in triplicate.
In vivo tumor xenograft assay
To generate tumor xenografts, EpCAM+CD44+HCT-116 cells (5×105) were injected subcutaneously into the right flank of each mouse. When the mice attained a tumor volume of 40-60 mm3, they were randomly divided into 3 groups (n = 10) and treated with Nanog siRNA, mock, or negative control siRNA. Nanog siRNA or negative control siRNA was injected intratumorally twice a week for 3 weeks. The tumor size was measured every other day using a vernier caliper and calculated as (a×b2)/2, where a is the tumor length and b the width. At the end of the experiment, mice were euthanized by carbon dioxide asphyxiation for approximately 6 min (air displacement rate: 20%/min; carbon dioxide flow rate: 1.7 L/min; the mortality was ensured by cervical dislocation) and tumors were excised and weighed.
All data are presented as the mean ± standard deviation (SD) of at least three repeat experiments. Student's t-test and one-way ANOVA analysis were used to analyze the variances between groups. The log-rank test was used to compare the survival rates in different groups. Significant differences were considered when P values were less than 0.05.