Patients and tissue samples
Primary CRC tissues and paired adjacent non-tumor mucosa tissues were obtained from 133 patients who underwent surgical resection at The First People's Hospital of Shangqiu (Shangqiu, China), and the clinicopathological characteristics of these patients were summarized in Table 1. None of these patients underwent chemotherapy or radiotherapy prior to surgery. After collection, all tissue samples were immediately snap-frozen in liquid nitrogen and stored at −80°C.
Cell culture and treatments
CRC cell lines (HCT116, HT29, SW620 and SW480) and human normal colon epithelial cells (FHC), purchased from American Type Culture Collection (ATCC; Manassas, VA, USA), were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA) and 1% penicillin/streptomycin at 37°C in a humidified incubator with 5% CO2.
Oxaliplatin (OXA) was purchased from Meilun Biological Co., Ltd. (Dalian, China). OXA-resistant CRC cell lines (HCT116/OR and SW480/OR cells) were established by exposure to incremental doses of OXA (up to 2 µM) for 3 months in our laboratory. OXA was removed before the experiments were performed.
The specific small interference RNA (siRNA) targeting CBR3-AS1 (si-CBR3-AS1), siRNA negative control (si-NC), miR-145-5p mimics, miR-145-5p inhibitor, miRNA mimics and inhibitor negative control were synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Cells were seeded into 6-well plates one day before transfection and then transfected with the aforementioned molecular products using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). 48 h post-transfection, the transfection efficiency was analyzed by RT-qPCR analysis.
Total RNA was extracted using Trizol reagent (Invitrogen), and quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.). Cytosolic and nuclear fractions of cells were isolated using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo Fisher Scientific, Inc.). cDNA was synthesized using the PrimeScript RT reagent Kit (TaKaRa, Dalian, China), and PCR amplifications were then carried out using a SYBR Green PCR Kit (TaKaRa) on a 7500HT Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Data were analyzed using 2−ΔΔCt method (7). GAPDH or U6 was used as an internal control.
Cells were seeded in 96-well plates at a density of 4,000 cells/well. Then, 20 µl MTT solution (5 mg/l; Sigma-Aldrich, St. Louis, MO, USA) was added to each well. Following incubation for additional 4 h, 150 μl DMSO (Sigma-Aldrich) was added to dissolve the formazan crystals. Then the absorbance of each well was recorded at 570 nm and read on a microplate reader (MultiskanEX, Lab systems, Helsinki, Finland).
Cell apoptosis analysis
Cells were resuspended by 500 μl 1× binding buffer, and then double-stained with 5 μl Annexin V-FITC and 5 μl PI using an Annexin-V-PI Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA). The stained cells were then analyzed using a flow cytometry (FACScan; BD Biosciences) equipped with CellQuest software.
Cells in serum-free medium were put into the uncoated or matrigel-coated upper chamber of transwell plates (8 μm pore size; Corning Inc., Corning, NY, USA), while 500 μl medium containing 10% FBS was added to the lower chamber. After 24 h, the cells that had not penetrated the membrane were removed using a cotton swab, while the cells on the lower membrane surface were fixed with 75% methanol and subsequently stained with 0.1% crystal violet. Then, the stained cells were captured using a microscope (Olympus, Tokyo, Japan).
Mammosphere formation assay
Single cells prepared by 0.25% trypsin-EDTA were seeded at a density of 2,000 cells/well in six-well ultra-low attachment plates (Corning Inc.), and then cultured with serum-free DMEM/F12 medium (Thermo Fisher Scientific, Inc.) containing 20 ng/ml human EGF, 1% B27 and 20 ng/ml fibroblast growth factor for 7 days. The number of mammospheres (>20 μm) was counted under an inverted microscope (Olympus).
Western blot analysis
Total protein was extracted using RIPA protein extraction reagent (Beyotime, Shanghai, China). Equal amounts of protein samples were separated by SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% skim milk powder for 2 h at room temperature, followed by incubation with the specific primary antibodies at 4°C overnight. The membranes were then incubated with HRP-conjugated secondary antibody at room temperature for 1 h. The blots were visualized using the enhanced chemiluminescence reagents (Bio-Rad Laboratories, Hercules, CA, USA). GAPDH was used as the loading control.
Dual-luciferase reporter assay
The fragment of CBR3-AS1 containing the predicted miR-145-5p-binding sites was synthesized and inserted into the psiCHECK-2 luciferase reporter vector (Promega, Madison, WI, USA), and mutations were achieved in the binding sites using a Mut Express II Fast Mutagenesis kit (Vazyme, Piscataway, NJ, USA). Cells were co-transfected with the luciferase constructs and miR-145-5p mimics or mimics negative control using Lipofectamine 2000. After 48 h of co-transfection, the luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega).
RNA immunoprecipitation (RIP) assay
RIP assay was performed using the EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore). The lyase sample of cells transfected with miR-145-5p mimics or mimics negative control was incubated with magnetic beads conjugated with anti-Ago2 or IgG antibody. The immunoprecipitated RNA was purified and subjected to RT-qPCR analysis.
All statistical analyses were performed using GraphPad Prism 6.0 software (GraphPad Software, Inc., La Jolla, CA, USA) and SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). The differences between groups were undertaken using Student’s t-test or one-way ANOVA followed by Tukey’s test. Survival curves were generated by Kaplan-Meier analysis and compared using log-rank test. All P values< 0.05 were considered to indicate statistical significance.