Tissues
Colorectal cancer tissues were obtained from patients at the First Affiliated Hospital of Bengbu Medical College. All the patients in the trial signed their consent forms, and all procedures were authorized by the Ethical Committee of the First Affiliated Hospital of Bengbu Medical College.
Immunohistochemistry
Paraffin-embedded CC tissues were de-paraffinized in xylene. The slides were blocked using 3% H2O2 for 15 min and then After blocking with 3% BSA for 60 minutes, the slides were immunostained overnight at 4°C with corresponding antibodies. The section was incubated with a secondary antibody the following day (1:1000 dilution; Abcam, CA, USA) and detected through a light microscope.
Cell Culture
Colorectal mucosal cells FHC cells and colorectal cancer cells HT29 and HCT116 obtained from BeNa Culture Collection (Hebei China) were cultured in RPMI 1640 medium with 10% FBS at 37°C in 5% CO2. Colorectal cancer cells were induced with 0, 5, 10, and 20ng/ml IL-17A (HY-P7372, MedChemExpress) for 24h, 48h, and 72h. JAK/STAT3 inhibitor AG490 (HY-12000, MedChemExpress) was used to induce the cell for 24h.
Rt-qpcr
Trizol was used to extract RNA from patient tumor tissue or HT29 and HCT116 cells. cDNA was synthesized using the Reverse Transcription Kit. The quantitative real-time PCR (qPCR) was operated with a Roche 480. A comparative 2−ΔΔCT was used to determine relative gene expression 12.
Western Blot
Total protein from patient tumor tissue or HT29 and HCT116 cells was isolated with lysis buffer (Beyotime). The BCA protein assay kit was then used to measure the protein concentrations. We separated the proteins using SDS-PAGE and transferred them to PVDF membranes (Millipore). 5% skimmed milk was used to block the membranes for 1 hour, followed by incubation with the primary antibodies at 4℃ for one night. The next day, the membranes were incubated using secondary antibodies. and visualized with an ECL kit, and analyzed using imaging software.
CCK8
HT29 and HCT116 cells were added into a 96-well plate (1×104 cells per well). After giving the corresponding treatment, each well added 10 µl of CCK8 solution (C0038; Beyotime) for 4 h and then measured the absorbance at 450 nm with a microplate reader.
Colony Formation Assay
HT29 and HCT116 cells were put in in 6-well plates (5000 cells per well) and then induced with IL-17A at a concentration of 10ng/ml. The cells were incubated for 14 days and then fixed with methanol, and stained with a 0.1% crystal violet solution for 30 minutes.
Cell Cycle Analysis Assay
Cells were put in 6-well plates (1×106 per well). Cells were fixed with 75% ethanol overnight at -20°C. The next day, a total of 450 µl PBS and 50 µl propidium iodide at a concentration of 0.5 mg/ml was added to each tube. The cell cycle was analyzed by flow cytometer.
Transwell Assay For Migration And Invasion
The treated HT29 and HCT116 cells were suspended (2.5 × 105 per well) in RPMI-1640 medium. Matrigel (BD Biosciences) was used for invasion assays. A 100 µl aliquot of the mixture was added to the upper chambers and incubated at 37oC for 5 h to solidify. 200 µl cell suspensions were added to the upper chamber, and 700 µl of cultured medium containing 10% FBS was added to the lower chamber. Fixation and staining of migrated cells with 4% paraformaldehyde and 0.1% crystal violet were performed for 20 minutes at room temperature after 24 h. Subsequently, the cells selected from five random fields were counted with a light microscope.
Statistical Analysis
Statistical significance between two groups was determined with a student’s t-test, and statistical significance between multiple groups was determined with one-way ANOVA using GraphPad Prism software version 8. Data were presented as means ± SD. P < 0.05 was considered to be statistically significant.