The human normal intestinal epithelial cell line NCM460 (BNCC353657) and the human colon cancer cell lines LoVo (BNCC338601) and SW480 (BNCC288146) were obtained from the BeNa Culture Collection (www.bnbio.com, Beijing, China) and cultured in RPMI-1640 (Gibco, Rockville, MD, USA) containing 10% foetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA). The cells were maintained in a humidified cell incubator (Thermo Fisher Scientific, Waltham, USA) atmosphere of 5% CO2 at 37°C.
Cell groups and transfection
LoVo and SW480 cells were divided into (1) blank control group (BC): no treatment; (2) miR-200c overexpression group: cells were transfected with 100 nM miR-200c mimic (5'-TCCATCATTACCCGGCAGTA-3') lentiviral vector; (3) fucosyltransferase 4 (FUT4) silencing group (si-FUT4): cells were transfected with 50 nM FUT4 siRNA (5'-GUUUGGAUGAACUUCGAGUTT-3', 5'-ACUCGAAGUUCAUCCAAACTT-3';) lentiviral vector; (4) miR-200c + FUT4 overexpression negative control group (miR-200c + NC1): cells were transfected with 100 nM miR-200c mimic and 50 nM FUT4 overexpression negative control lentiviral vector; (5) miR-200c + FUT4 overexpression negative control group (miR-200c + FUT4): cells were transfected with 100 nM miR-200c mimic and 50 nM FUT4 mimic viral vector. In each group, there were three replicates.
LoVo and SW480 cells in logarithmic growth phase were selected for subsequent experiments. The cells were passaged 1 day before transfection and cultured in a 6-well plate. When the confluence reached 70%, transfection was performed according to the lentiviral transfection instructions. Lentiviral particles were constructed by Shanghai Jikai Biotechnology Co., Ltd. (Shanghai, China). MiR-200c mimic, FUT4 siRNA, FUT4 mimic and negative control lentiviral vectors were purchased from Shanghai GenePharm Pharmaceutical Technology Co., Ltd. (Shanghai, China). The expression of miR-200c and FUT4 mRNA in transfected cells was detected by real-time quantitative polymerase chain reaction (RT-qPCR) at 72 h after transfection. Each experiment was repeated three times.
A total RNA extraction kit (A27828, MagMAX ™ MiRVana ™ Total RNA Isolation Kit, Thermo Fisher Scientific, Waltham, USA) was used to extract total RNA from the cells. cDNA was synthesized by a reverse transcription kit (Applied Biosystems, Waltham, MA, USA), and SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA USA) was used for RT-qPCR. The miR-200c primer was synthesized by Shanghai Shengong Biotechnology Co., Ltd. (Shanghai, China), and the reaction was performed under the following conditions (40 cycles): 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 1 min. The miR-200c and FUT4 mRNA compared with the endogenous controls U6 and GAPDH, respectively, and the data were processed by the 2-ΔΔCt method. The sequences of the primers were as follows: miR-200c, forward: 5'-CCTATGTAAACAGCCTCGACTG-3' and reverse: 5'-CTGGCGTATCGTGAGTCG-3'. U6, forward: 5'-GACCTCTATGCCAACACAGT-3' and reverse: 5'-AGTACTTGCGCTCAGGAGGA-3'. FUT4, forward: 5'-AAGGTCCAGGCCCACTGAAG-3' and reverse: 5'-CAGTTCAGGTGACAGAGGCTCA-3'. GAPDH, forward: 5'-ATGGGGAAGGTGAAGGTCG-3' and reverse: 5'-GGGGTCATTGATGGCAACAATA-3'.
Double luciferase reporter assay
Target gene prediction between miR-200c and FUT4 was performed using TargetScan software (www.targetscan.org). Wild-type and mutant 3'-UTRs of FUT4 were amplified in the pGL3/luciferase vector (Promega, Madison, WI, USA) and cloned downstream of the luciferase gene. The constructed luciferase reporter plasmid (wt-FUT4 or mut-FUT4) was separately co-transfected with miR-200c or NC into LoVo and SW480 cells using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, China) for 24 h. The luciferase activity of the cells was detected with the dual luciferase reporter system (Promega) at 48 h after transfection according to the instructions.
Cell Counting Kit (CCK)-8 assay
Cells at logarithmic growth phase were plated into 96-well plates at a density of 2×104 cells/mL, 100 μL per well. According to the manufacturer’s instructions (G021-1-1, Nanjing Jiancheng Bioengineering Institute, China), cells viabilities were analyzed. The optical density (OD) of each well at 450 nm was measured by a microplate reader.
Plate colony formation assay
Logarithmic growth phase cells were digested with 0.25% trypsin and adjusted to 250 cells/mL. Cells (2 mL/well) were cultured in a 6-well plate at 37 °C and 5% CO2 for 2-3 weeks, and the fresh medium was added every 3 days. Methanol was used to fix the cells, and 1 ml of Ji Giemsa working fluid was used to stain the cells for 30 min. After two washes with ultrapure water, filter paper was used to remove the water around the dish, and the cells were imaged by a camera.
Transwell assay to analyse cell migration and invasion
Cell invasion experiment: After digestion, centrifugation and resuspension, the cells were adjusted to 4 × 105 cells/mL. Fifty microlitres of 1640 medium containing Matrigel (1:1) without FBS was added to the transwell upper chamber and incubated at 37 °C for 1 h. Then, 100 μL of cell suspension was added to the upper compartment of the chamber, while 600 µL of complete medium containing 10% FBS was added to the lower chamber. After incubation at 37 °C and 5% CO2 for 24 h, the membranes were fixed with methanol for 30 min and stained with crystal violet for 15 min. The non-migrated cells in the upper layer were gently wiped with cotton swabs. The results were observed under a light microscope (Olympus Corporation) and assessed by ImageJ software.
For the cell migration experiment, Matrigel was not required, and the other experimental steps were the same as those for the invasion experiment.
The cells in coverslips were treated differently as required and fixed with 4% paraformaldehyde. With 0.2% Triton X-100 cell permeabilization, the cells were blocked with 5% bovine serum albumin (BSA) and incubated in an incubator for 30 min at 37°C. Then, the cells were incubated with primary antibodies against Ki67 (1:200, orb88614, Biorbyt, Cambridge, UK) at 4 °C overnight. After the cells were rinsed with phosphate-buffered saline (PBS), they were incubated with FITC-labelled secondary antibody at 37 °C for 30 min in the dark. Subsequently, the cells were rinsed with PBS, stained with 4',6-diamidino-2-phenylindole (DAPI) and mounted with glycerol. The fluorescence was observed under an inverted laser confocal microscope (FV1200; New Discovery Technology (China) Co., Ltd, Shanghai, China).
The cells or tumor tissues were split using lysozyme solution (90082, ThermoFisher, Shanghai, China), and the protein concentration of the cells was measured using a BCA kit (Solarbio, Beijing, China). Then, the protein samples were transferred to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis (Mini-Protean-3, Bio-Rad, Hercules, CA, USA) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Massachusetts, USA). After the membranes were blocked with 5% skim milk, they were incubated with primary rabbit anti-human antibodies against β-catenin (1:2000, ab16051, Abcam), CyclinD1 (1:200, ab16663, Abcam), GSK-3β (1:5000, ab32391, Abcam), p-GSK-3β (1:1000, ab131097, Abcam), and β-actin(1:2500, ab8227, Abcam, UK) and mouse anti-human FUT4 (1:1000, sc-19648, Santa Cruz Biotechnology, Beijing, China). After three washes with TBST (TBS, 1 ml/L Tween-20), the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit (1:2000, ab6721, Abcam) and goat anti-mouse (1:2000, sc-2354, Santa Cruz Biotechnology) immunoglobulin G secondary antibodies. Finally, the enhanced chemiluminescence (ECL) method was used for detecting signals, and greyscale scanning and quantification of the protein bands were performed by ImageJ (NIH) software. The expression levels of proteins were normalized to β-actin.
Thirty SPF grade BALB/C female nude mice, body weight 16-18 g, 4 weeks old, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (license no. scxk (Jing) 20160006). The animals were raised at 26-28 °C and a humidity of 40% - 60%. The feed, drinking water and bedding materials were sterilized. Animal experiments followed the national institutes of health guidelines (NIH pub. no. 85-23, revised 1996) and were approved by the Animal Protection and Use Committee of Shengjing Hospital.
Xenograft tumour model
Thirty mice were randomly divided into 5 groups (n = 6): the model group: mice were subcutaneously injected with 200 μL of normal saline; the miR-200c group: mice were subcutaneously injected with 200 μL (5 × 106 cells/100 μL) of LoVo and SW480 cells , which were transfected with miR-200c mimic; the si-FUT4 group: mice were subcutaneously injected with 200 μL (5 × 106 cells/100 μL) of LoVo and SW480 cells, which were transfected with siRNA FUT4 lentiviral vector; the miR-200c + NC1 group: mice were subcutaneously injected with 200 μL (5 × 106 cells/100 μL) of LoVo and SW480 cells, which were transfected with miR-200c mimic and FUT4 negative control; the miR-200c + FUT4 group: mice were subcutaneously injected with 200 μL (5 × 106 cells/100 μL) of LoVo and SW480 cells, which were simultaneously transfected with miR-200c mimic and FUT4 mimic.
The long diameter (L) and short diameter (W) of the tumour were measured every 7 days, and the tumour volume was calculated. Tumour volume (V) = (long diameter × short diameter 2)/2. After 28 days, the nude mice were anaesthetized by intraperitoneal injection of 3% pentobarbital sodium (40 mg/kg). The tumour tissues were weighed. The positive expression of Ki-67 was detected by immunohistochemistry and immunofluorescence staining.
The tumour tissue was heated, dewaxed with xylene, and then hydrated with gradient ethanol solution. A 3% H2O2 methanol solution was added for inactivation for 20 min, high temperature antigen in citrate buffer solution (pH 6.0) was used for thermal repair for 10 min, and 5% BSA was used for blocking treatment for 20 min. Rabbit anti-human Ki67 (1:200, ab15580, Abcam) polyclonal antibody was added and incubated overnight at 4 °C. After rewarming, goat anti-rabbit IgG labelled with horseradish peroxidase (1:1000, abin101988, Antibodies Online, Germany) was incubated with the secondary antibody, and DAB staining was performed. The cells were observed under an optical microscope at 400× magnification (Olympus, Japan). The results are expressed as the percentage of positive cells (%).
SPSS 19.0 statistical analysis software was used for data processing, and the results of data analysis are expressed as the mean ± standard deviation (mean ± SD). The t-test was used for data analysis between two groups, and one-way analysis of variance (ANOVA) with Dunnett’s t test was used for data analysis of multiple-group comparisons. The difference was statistically significant at p < 0.05.