Cell culture and transfection
HCT116 cells and HT29 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), and cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium (HyClone, South Logan, UT, USA) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), 100 U/mL penicillin and 100 μg/mL streptomycin. HCT116/DDP and HT29/DDP cells were induced in our laboratory and cultured in RPMI-1640 medium with 20 μg/mL cisplatin, respectively. Cells that were logarithmically grown and in good condition were inoculated into 6 well plates before transfection with 1×105 cells/well. Cell abundance during transfection was 60-70%. Transfection was performed for 48 h using Lipo fectamineTM 2000.
RNA extraction and real time polymerase chain reaction (RT-PCR)
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and quantified using UV spectrophotometer. D260 nm/D280 nm value was calculated for selecting qualified RNA samples, which were preserved at -20°C for use. Extracted RNA was reversely transcribed into cDNA and amplified by Real Time quantitative Polymerase Chain Reaction (RT-qPCR) using SYBR Premix Ex Taq II kit (TaKaRa, Otsu, Shiga, Japan). PCR reaction conditions were: pre-denaturation at 95°C for 30 s, 95°C for 5 s, and 60°C for 31 s, for a total of 40 cycles. The relative levels were quantitatively analyzed using the 2-ΔΔCt method. GAPDH was used as an internal reference. The experiment was repeated for three times.
Apoptosis determination
Cells collected were washed twice with phosphate-buffered saline (PBS) and stained using the Annexin V/fluorescein isothiocyanate (FITC) Apop-tosis Detection Kit (Beyotime, Shanghai, China). The single cell suspension was first incubated with 500 μL of 1×buffer, 5 μL of Annexin V and 5 μL of Propidium Iodide (PI) at 37°C for 25 min in the dark. The above cells were examined by FACSAriaTM flow cytometry and analyzed by Win MDI. 2.9 software (TSRI Flow Cytometry Core Facility, La Jolla, CA). Every experiment was repeated three times.
Western blot
Total protein from cells was extracted with RIPA Lysis Buffer (RIPA; Beyotime, Shanghai, China). The protein were quantified by bicinchoninic acid (BCA) method (Pierce, Waltham, MA, USA) and loaded for electrophoresis. After transferring on a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA), it was blocked in 5% skim milk for 2 hours, incubated with primary antibodies at 4°C overnight and secondary antibodies for 2 h. Bands were exposed by enhanced chemiluminescence (ECL) and analyzed by Image J Software (NIH, Bethesda, MD, USA).
Xenograft animal experiments
six weeks old nude mice were bought from Beijing HFK Bioscience (Beijing, China). The mices were randomized into four groups (n = 6 per group). The cells were injected subcutaneously into the right dorsal flanks of the mice at 6 × 106 cells per animal. The tumor sizes were measured every three days using a caliper. Tumor size was calculated according to the following formula: V (mm3) = length × width2/2. The mice were killed using CO2 inhalation followed by cervical dislocation. The animal experiments were performed according to the institutional guidelines for animal care and approved by the Institutional Animal Care and Use Committee of the Chinese Academy of Medical Sciences.
Statistical analysis
Statistical analyses were performed using SPSS software (version 13.0). The t-test was used for analyzing the differences between the two groups. p<0.05 indicated the significant difference.