Patients’ samples collection
Fifty-four paired CRC tissues and adjacent non-tumor tissues were collected from March, 2015 to May, 2019 in Tangdu Hospital, the Air Force Medical University. Informed consent was provided for all CRC patients or their guardians and the study was permitted by the Ethics Committee of Tangdu Hospital, the Air Force Medical University.
Cell culture and transfection
We obtained four CRC cell lines (including SW620, SW480, HT-29, and HCT-116) and the normal colon epithelial cell line FHC from Shanghai Institute of Biochemistry and Cell biology (Shanghai, China). The cells were cultured in DMEM, (Gibco, USA) with 10% FBS (Tianhang, Hangzhou, China) and 1% penicillin-streptomycin in a humidified incubator (37 °C, 5% CO2). The overexpression vector of miR-497-5p (miR-497-5p) and the silence vector of miR-497-5p (miR-497-5p inhibitor) as well as NC mimic or NC inhibitor (the corresponding negative control), shRNA targeting XIST (si-XIST), shRNA scramble control (si-NC), pcDNA3.1-FOXK1 overexpression vector (FOXK1), pcDNA3.1 empty vector (pcDNA) were bought from Ribobio (Guangzhou, China) and transfected into HT29 and SW480 cells using Lipofectamine 3000.
Cell proliferation assays
The viability of HT29 and SW480 cells was evaluated by the Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) assay. The HT29 and SW480 cells (1 × 103cells/ml per 96-well) cultured for 24, 48, and 72 h, respectively. For cell proliferation assay, each well was added 10 µl CCK-8 solution for 2 h. The absorbance was detected at 450 nm using the microplate reader.
Flow cytometric analysis of apoptosis
HT29 and SW480 cells were harvested and fixed in pre-cooled ethanol, then resuspended in cold buffer with 5 µl of Annexin V-FITC. The samples were then incubated for 5 min after add 5 µl of PI and 200 µl of binding buffer. Cell apoptosis were analyzed using Annexin FITC/PI flow cytometry assay kit.
Cell Migration and Invasion Assay
Transwell assay was conducted to explore the cell migration and invasion. The upper chamber with Matrigel coating (8.0 µm PET membrane, 24 well plate, Corning, USA). The cells were placed into upper chamber with 1 × 104 cells/well and cultured in 400µL serum-free DMEM medium. Then 600 µL DMEM with 10% FBS was added into the lower chamber. After incubation for 24 h, cells on the bottom of the upper chamber was fixed with 90% ethanol solution for 30 min. 0.1% crystal violet was used to stain the cells for 10 min. At last, the invasion cells were observed by the light microscope (Olympus, Japan).
Dual-luciferase reporter assay
The sequence of XIST or FOXK1containing a miR-497-5p binding site was amplified and cloned to psiCHECK-2 vector (Promega, Madison, WI, USA) to generate XIST-WT (wild-type) or FOXK1-WT (wild-type). The binding site of XIST was mutated to obtain the XIST-MUT (mutant type) or FOXK1-MUT (mutant type) using a Site-Directed Mutagenesis Kit (Stratagene, California, USA). XIST-WT (or XIST-MUT), FOXK1-WT (or FOXK1-MUT) and miR-497-5p mimic were co-transfected into HT-29 and SW480 cells. And Dual-Luciferase Reporter Assay System (Promega, Madison, USA) was implemented to determine the luciferase activities.
RNA isolation and quantitative reverse transcription polymerase (qRT-PCR)
Total RNA extraction was conducted using Trizol Reagent (Shanghai Pufei Biotech Co., Ltd., Shanghai, China). cDNA was obtained by reverse transcription after DNA elimination. The prepared cDNA was amplified using SYBR Green Master Mixture (Takara, Otsu, Japan), of which the results were calculated by LightCycler® 480 real-time PCR system (Roche, Indianapolis, Ind). The thermocycling conditions were applied as follows: DNA regeneration at 95 °C for 5 min, 40 cycles at 95 °C for 30 sec, followed by primer annealing at 60 °C for 30 sec and primer extension at 72 °C for 5 min.
Western blot
The RIPA lysis buffer containg protease inhibitor firstly used to lyse the cells or tissues. The total protein was isolated from cell or tissue lysates after centrifugation. The concentration of protein was assessed using a Bradford Protein Assay Kit (Beyotime, China). Then 30 µg protein was separated by an SDS-PAGE and electro-transferred onto a polyvinylidene fluoride (PVDF) membranes (Millipore, Boston, MA, USA) (250 mA, 2 h), blocked with 5% skim milk for 1 h and then incubated with the primary antibodies: anit-FOXK1 (1: 1000 dilution), and anti-β-actin primary antibodies (1: 5000 dilution, Cell Signaling Technology, USA), at 4 °C overnight, followed by incubation with the secondary antibodies for at room temperature for 1 h. Finally, the immune bans were detected using the enhanced chemiluminescence system.
Xenograft Tumor Model
Male BALB/c nude mice (about aging 4 weeks) were purchased from Charles River (Beijing, China) and further used for the xenograft assays. All animal experimental procedures were approved by the Ethics Committee for Animal Studies of Tangdu Hospital, the Air Force Medical University. HT29 cells (2 × 106) transfected with si-XIST or si-NC were injected subcutaneously into one flank of every nude mouse. And then the tumor sizes were measured every four days followed the calculated tumor volumes. And all mice were killed after 24 days, and tumor masses were weighted and used for subsequent molecular analysis
Statistical analysis
All data of this study were expressed as the mean ± standard deviation (SD) and repeated at least three times. SPSS 22.0 software was used to conduct all statistical analyses (SPSS, Inc, USA). The comparison between the data of the groups was analyzed by the Student’s t-test and two-way analysis of variance. The p-vale < 0.05 indicated statistical significance.