Cell lines and reagents
Human colorectal carcinoma cell lines HCT116, HCT8, and HT29 were purchased from the American Type Culture Collection (Manassas, VA, USA). These cells were cultured in RPMI 1640 media (Corning Incorporated, Corning, NY, USA) containing 10 % FBS (RMBIO, Missoula, MT, USA), glutamine, and antibiotics and incubated at 37 °C in 5 % CO2. Lipopolysaccharide (LPS, a TLR4 ligand), flagellin (a TLR5 ligand), Ox, 5-Fu, and fibronectin were purchased from Sigma-Aldrich (St. Louis, MO, USA). FSL-1 (a TLR2 and TLR6 ligand) was obtained from Invivogen (San Diego, CA, USA). Sp1 miScript target protector for miR-125b was purchased from Qiagen (Hilden, Germany). Bay 11-7082 (an NF-κB inhibitor) was obtained from Selleckchem (Houston, TX, USA).
Cytotoxicity assay for IC50 determination and establishment of drug-resistant cell lines
Cells (2x104 cells/well) in 96-well plates were exposed to various concentrations of Ox or 5-Fu for 24 h. The inhibition rate of cell growth was measured using the following formula: inhibition rate (%) = [1 - OD570 (experiment group)/OD570 (control group)] × 100 [23]. Cytotoxicity curves were obtained using GraphPad software by plotting the measured cell viability (%) indicated by Cell Counting Kit-8 (CCK-8) assays (Sigma‑Aldrich; Merck KGaA). Based on the CCK-8 assay results, the 50 % inhibitory concentration (IC50) values for Ox and 5-Fu were evaluated and calculated (Supplemental Table 1). Next, to create colorectal cancer cell lines with stable chronic resistance to Ox or 5-Fu, HCT116, HCT8, and HT29 cells were exposed to an initial concentration of Ox or 5-Fu at 1 μM in RPMI 1640 medium plus 10 % FBS, as previously described with slight modifications [24]. When cells that survived Ox or 5-Fu treatment reached 70~90 % confluency, they were subcultured twice a week to confirm their viability. Thereafter, the dose of Ox or 5-Fu was doubled in each surviving population and sequentially increased to 50 μM. All resistant cell lines maintained and experimented at 20 μM Ox or 5-Fu in RPMI 1640 medium plus 10 % FBS. Finally, the authenticity of the drug-resistant sublines was verified by short tandem repeat profiling according to the ANSI Standard (ASN-0002) from the ATCC Standards Development Organization (SDO).
MicroRNA microarray assay
Total RNA from Oxaliplatin- or 5-fluorouracil-resistant HCT8 was separated using the miRNeasy Mini Kit (Qiagen, CA, USA). RNA purity and integrity for analysis were measured by ND-1000 Spectrophotometer (NanoDrop, Wilmington, USA), Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, USA). The Affymetrix Genechip miRNA 4.0 array process was executed according to the manufacturer's protocol and previously described [25]. Briefly, biotin-tagged RNA of each sample by FlashTag™ Biotin RNA Labeling Kit (Genisphere, Hatfield, PA, USA) was quantified, fractionated, and hybridized according to the standard procedures provided by the manufacture. After RNA-array hybridization, the chips were washed, stained, and then scanned with an Affymetrix GCS 3000 canner (Affymetrix, Santa Clara, California, United States). Signal values were compare and calculated with using the Affymetrix® GeneChip™ Command Console software (AGCC).
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA from cells was extracted using an RNeasy Mini Kit (Qiagen) according to the supplier’s instructions. cDNA was synthesized from 2 μg of purified total RNA using Accupower® RT premix (Bioneer, Daejeon, Korea) and an oligo(dT) primer (Bioneer, Daejeon, Korea). To evaluate miRNA levels, total RNA was isolated from cells using an miRNeasy Mini Kit (Qiagen). cDNA was synthesized with a Mir-XTM miRNA First-Strand Synthesis Kit (Clontech, Mountain View, CA, USA). The mRNA and miRNA levels were quantified using SYBR Green (Takara, Tokyo, Japan), an ABI7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA), and specific primer sets for TLR2 (upstream primer, 5’-GGC CAG CAA ATT ACC TGT GTG; downstream primer, 5’-CCA GGT AGG TCT TGG TGT TCA), TLR4 (upstream primer, 5’-CTG CAA TGG ATC AAG GAC CA; downstream primer, 5’-TCC CAC TCC AGG TAA GTG TT), TLR5 (upstream primer, 5’-CAT TGT ATG CAC TGT CAC TC; downstream primer, 5’-CCA CCA CCA TGA TGA GAG CA), TLR6 (upstream primer, 5’-TAG GTC TCA TGA CGA AGG AT; downstream primer, 5’-GGC CAC TGC AAA TAA CTC CG), CD248 (upstream primer, 5’-TGG TGC CAA CGT GTG TCT TTT; downstream primer, 5’-AGC GAT AGC AGT CAG TGA TGC), Sp1 (upstream primer, 5’-GCG AGA GGC CAT TTA TGT GT; downstream primer, 5’-GGC CTC CCT TCT TAT TCT GG), and miR-125b-5p (5’-TCC CTG AGA CCC TAA CTT GTG A). A specific primer set for β-actin (upstream primer, 5’-ATC CAC GAA ACT ACC TTC AA; downstream primer, 5’-ATC CAC ACG GAG TAC TTG C) was used as internal control for mRNA, and U6 from the Mir-XTM miRNA First-Strand Synthesis Kit was used as a control for miRNA.
RNA-binding Protein Immunoprecipitation (RIP) assay for miRNA
The RIP assay kit for miRNA (MBL, Nagoya, Japan) was used to confirm between miR-125b-5p and Sp1 interaction by following the supplier’s protocol. Briefly, anti-EIF2C2/AGO2 mouse monoclonal antibody (MBL) or mouse IgG2a isotype control (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) were incubated with protein G beads (Santa Cruz Biotechnology, CA, USA) at 4 °C for 4 h. The fresh cell extracts were immunoprecipitated with the antibody-immobilized protein G beads at 4 °C overnight. The immunoprecipitated protein were eluted by boiling with Laemmli sample buffer. The immunoprecipitated RNAs were seperated using the two-step method described in the supplier’s recommendations. Sp1 and miR-125b-5p expression levels were examined after total RNA isolation from antibody-immobilized Protein G agarose bead-ribonucleoprotein (RNP) complexes by real-time PCR.
Western blot analysis
Harvested cells were lysed with NP-40 buffer (Elpis Biotech, Daejeon, Korea) supplemented with a protease inhibitor cocktail and phosphatase inhibitors (Sigma-Aldrich). Equally quantified proteins (10 μg/sample) according to the results from a BCA assay kit (Pierce, Rockford, IL, USA) were subsequently loaded onto SDS-PAGE gels. After electrophoresis, the proteins were transferred onto NC membranes (Millipore Corp., Billerica, MA, USA). The membranes were then blocked with 5 % non-fat skim milk and probed with primary antibodies. The expression levels of the target proteins were determined using a chemiluminescence kit (Advansta Corp., Menlo Park, CA, USA) and an Amersham Imager 600 (GE Healthcare Life Sciences, Little Chalfont, UK). The expression levels of β-actin were established as a control.
Small interfering RNA (siRNA) or micro RNA (miRNA) transfection
Human TLR2-siRNA (5’-GGG CAG UCU UGA ACA UUU AUU-3’), TLR6-siRNA (5’-CCA GAA UCC AGU UCU CCG A-3’), TLR5-siRNA (5’-CUC GCU UGG AUC UAU CCA-3’), CD248-siRNA (5’-GCG AAC ACG AAU GUG UGG A-3’), Sp1-siRNA (5’-CAG AUA CCA GAC CUC UUC U-3’), and negative control siRNA (Cat. No. SN-1001-CFG) were purchased from Bioneer. A mature miR-125b mimic and negative control miRNA mimic were designed and synthesized by Bioneer. The sequence of the miR-125b-5p mimic was 5’-UCC CUG AGA CCC UAA CUU GUG-3’, and the sequence of the miR-125b-5p inhibitor was 5’-UCC CUG AGA CCC UAA CUU GUG A-3’. Specific primers for the negative control miRNA mimic (Cat. No. SMC-2001) and negative control miRNA inhibitor (Cat. No. SMC-2101) were purchased from Bioneer. Transfection with 200 nM siRNA or 200 nM miRNA using Lipofectamine RNAiMAX Reagent (Invitrogen, Carlsbad, CA, USA) was performed according to the supplier’s instructions. The cells were used for further experiments at 48 h after transfection.
Scratch assay and migration assay for detecting cancer cell invasion activity
Cells were seeded onto a 6-well plate and allowed to grow to approximately 90 % confluency. The cells were wounded with a 200-μl plastic tip and then incubated in 5 % CO2 at 37 °C for 24 h. After 24 h incubation, the cells were checked, and images were taken under an inverted phase contrast microscope equipped with a camera at ×100 magnification. According to the supplier’s instructions, the migratory and invasive activities of cancer cells were determined using a CytoSelectTM tumor transendothelial migration assay kit (Cell Biolabs, Inc., San Diego, CA, USA) and a CultreCoat 96-well Medium BME Cell Invasion Assay Kit (R&D Systems, Minneapolis, MN, USA), respectively. The relative fluorescence units (RFUs) of migrated cells were determined by a microplate reader. The relative invasion of cells was compared with the fluorescence intensity of calcein-AM stained invading cells measured by a microplate reader.
Measurement of NF-κB translocation and NF-κB DNA-binding activity
Nuclear and cytoplasmic fractions were prepared using a Nuclear/Cytosol Fractionation Kit (BioVision Inc., Mountain View, CA, USA) according to the instructions of the supplier. Briefly, harvested cells (2 × 106/sample) were resuspended in cytosol extraction buffer A and incubated for 10 min on ice. After mixing with cytosol extraction buffer B, the supernatants collected by centrifugation were designated cytosolic fractions, and the pellets were suspended in 100 μl of nuclear extraction buffer mix and designated nuclear fractions. All samples were kept at -80 °C until use. To block NF-κB activity, cells were treated with Bay11-7082 (an NF-κB inhibitor; 5 μM) for 8 h or 24 h before the designated experiments. The DNA-binding activity of NF-κB was measured as described previously [26] using an NF-κB p50/p65 Transcription Factor Assay Kit (Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions.
Quantification of human cytokines by ELISA
The concentrations of EMT-related cytokines (TGF-β1, TNF-α, VEGF, and IL-8) in the cell culture supernatants were measured as described previously [27] using single cytokine ELISA assay kits (R&D Systems) according to the supplier’s instructions.
Analysis of apoptosis by flow cytometry
The percentages of cells undergoing the apoptotic process were measured by flow cytometry with fluorescein isothiocyanate (FITC)-labeled annexin-V (BD Biosciences, San Diego, CA, USA) and 7-amino actinomycin D (7-AAD) (BD Biosciences). The cells were suspended in 100 μl of 1× annexin-V binding buffer, and FITC-conjugated annexin-V (3 μl) and 7-AAD (3 μl) were then added to the suspensions; the cells were kept at room temperature for 15 min in the dark. Finally, the stained cells were monitored by a BD AccuriTM C6 (BD Biosciences).
Statistical analysis
One-way analysis of variance (ANOVA) using SPSS version 24.0 statistical software (IBM Corp., Armonk, NY, USA) or Student’s t-test was used for all statistical analysis. Data are presented as the mean ± standard deviation (SD). Differences were determined to be statistically significant at p<0.05 and highly significant at p<0.01.