Cell culture
Human normal intestinal epithelial cells NCM460 (BNCC353657), human colon cancer cells Lovo (BNCC338601) and SW480 (BNCC288146) were selected and cultured in RPMI-1640 (Gibco, Rockville, MD, USA) containing 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA). The cells were all obtained from Shanghai Institute of Cell Research, CAS. The cells were maintained in a humidified cell incubator (Thermo Fisher Scientific, Waltham, USA) atmosphere of 5% CO2 at 37ºC.
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 lentiviral vector;17 (3) fucosyltransferase 4 (FUT4) silencing group (si-FUT4): cells were transfected with 50 nM FUT4 siRNA lentiviral vector;17 (4) miR-200c + FUT4 overexpression negative control group (miR-200c + NC1): cells were transfected with 100 nM miR-200c overexpression lentiviral vector and 50 nM FUT4 overexpression negative control lentiviral vector; (5) miR-200c + FUT4 overexpression group (miR-200c + FUT4): cells were transfected with 100 nM miR-200c overexpression lentiviral vector and 50 nM FUT4 overexpression lentiviral vectors.
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, mimic-NC, FUT4 siRNA, FUT4 mimic, and FUT4 overexpression 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 quantitative real-time polymerase chain reaction (qRT-PCR) at 72 h after infection.
Cell counting kit (CCK)-8 assay
Logarithmic growth phase cells were inoculated into 96-well plates at a density of 2 × 104 cells/ml, 100 µl per well. After cultured for 24 h, 48 h, 72 h, and 96 h at 37ºC, 5% CO2 in incubator, 10 µl of CCK-8 solution (Tongren Institute of Chemistry, Japan) was added into each well and incubation for another 4 h. The optical density (OD) of each well at 450 nm was measured by a microplate reader.
Colony formation assay
Logarithmic growth phase cells were digested with 0.25% trypsin and adjusted to 250 cells/ml. 2 ml/well cells were cultured in a 6-well plate at 37 °C, 5% CO2 for 2–3 weeks and the fresh medium was changed every 3 days. Methanol was used to fixed the cells and 1 ml of Ji Giemsa working fluid was used to stain the cells for 30 min. After washed twice with ultrapure water, the filter paper was used to suck up the water around the dish and the record was imaged by a camera.
Transwell assay
Cell invasion experiment: After digesting, centrifuging and resuspending, the cells were adjusted to 4 × 105 cells/ml. 50 µl of 1640 medium containing Matrigel (1:1) were added to the transwell upper chamber, and incubated at 37 °C for 1 h. Then, 100 µl cell suspension was added into the upper compartment of the chamber while 600 ul 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 results were observed under a light microscope (Olympus Corporation) and performed by ImageJ software.
For cell migration experiment, matrigel is not required, and other experimental steps were the same as invasion experiment.
Immunofluorescence
The cells crawling in coverslips were treated differently as required and fixed with 4% paraformaldehyde. After rupturing the membrane with 0.2% Triton X-100, cells were sealed with 5% bovine serum albumin (BSA) and incubated in incubator for 30 min. Then, the cells were incubated with primary antibodies Ki67 (1:200, orb88614, Biorbyt, Cambridge, UK) at 4 °C overnight. After rinsing with phosphate buffer saline (PBS), the cells were incubated with FITC-labeled 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 the glycerol. The fluorescence was observed under an inverted laser confocal microscope (FV1200; New Discovery Technology (China) Co., Ltd, Shanghai, China).
qRT-PCR
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 the reverse transcription kit (Applied Biosystems, Waltham, MA, USA) and SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA USA) was used for RT-PCR. 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 were internally referenced with U6 and GAPDH, respectively, and the data was processed by the 2−ΔΔCt method. The sequence of the primers are 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'.
Western blot
After lysed and centrifuged, the protein concentration of the cells was measured by BCA kit (Solarbio, Beijing, China). Then, the protein samples were transferred to sodium dodecyl sulfate-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 blocking with 5% skim milk, the membranes 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), GAPDH (1:2500, ab9485, Abcam, UK), and mouse anti-human FUT4 (1:1000, sc-19648, Santa Cruz Biotechnology). After washed three times with TBST (TBS, 1 ml/L Tween-20), the members 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) chemiluminescence method was used for detecting signals, and the grayscale scanning and quantification of the protein band were performed by Image J (NIH) software. The expression levels of proteins were normalized to GAPDH.
Double luciferase reporter assay
Wild type and mutant 3'UTRs of FUT4 were amplified in pGL3/luciferase vector (Promega, Madison, WI, USA) and cloned downstream of the luciferase gene. The luciferase activity of the cells was detected with the dual luciferase reporter system (Promega) at 48 h after transfection according to the instructions.
Statistical analysis
SPSS 19.0 statistical analysis software was used for data processing, and the results of data analysis were expressed as mean ± standard deviation (mean ± SD). The t-test was exerted for data analysis between two groups, and one-way analysis of variance (ANOVA) was used for data analysis of multiple-group comparison. The difference was statistically significant at p < 0.05.