Cell cultures and reagents
HCT116, LoVo, SW620, SW480, HT-29, and HCT-8 human CRC cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). HCT116/HT-29 cells were preserved in McCoy's 5A medium, HCT-8 cells were preserved in 1640 medium, LoVo cells were preserved in F-12K medium (Kaighn's Modification of Ham's F-12 Medium), and SW480/SW620 cells were preserved in Leibovitz's L-15 medium. The media were supplemented with 10% fetal bovine serum, and cells were incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C.
HCT116 GR (gefitinib-resistant), LoVo GR, SW620 GR, and HT-29 GR cells in the logarithmic growth phase (convergence degree 60%-80%) were added with gefitinib (ZD1839) with an initial concentration of 1/10 of the parent cell line IC50 for 24h. The culture medium was discarded, washed twice with PBS, and the drug-free medium was replaced. After cell growth resumed, the cells were treated with low concentration for 24h. After the cells proliferate to normal morphology, the above drug shock was repeated, with 6-8 times of shock at each concentration. After the cells grew stably at this concentration, the culture was continued by increasing the concentration of drugs by 1.2-1.5 times. Drug induction lasted for 6 months until the cells were able to grow steadily in the concentration of the drug.
Lentiviruses containing miR-323a or control were purchased from GeneChem (Shanghai, China), and the transfection of lentivirus into CRC cells was performed according to the manufacturer’s protocol. After lentivirus infection, monoclonal cells were selected and cultured to check for expression of miR-323a-3p/5p by qRT-PCR. MicroRNA mimics were synthesized by RiboBio (Guangzhou, China) and transfected into CRC cells using Lipofectamine™ 2000 (Invitrogen, USA) according to the manufacturer’s protocol.
Receptor tyrosine kinase phosphorylation profile
To investigate the activation/phosphorylation of RTKs, we used the Human RTK Phosphorylation Antibody Array Membrane (ab193662, Abcam). The human phospho-RTK antibody array is a nitrocellulose membrane with 71 different anti-RTK antibodies spotted in duplicate on it, including 4 positive and 3 negative controls and 1 blank.
To perform a proteome profiler array experiment, cell lysates were prepared from GR or NC cells using Cell Lysis Buffer supplemented with Phosphatase Inhibitor and Protease Inhibitor Cocktail and stored at −80°C until use. For each cell lysate, 200 μg of total protein (determined by the Pierce BCA Protein Assay (Fisher Scientific) was diluted 1:5 with blocking buffer, placed onto each membrane, and incubated overnight at 4°C (16 hours). The antibody array membranes were washed and subsequently incubated with a biotinylated anti-phosphotyrosine antibody overnight at 4°C to detect phosphorylated tyrosine on activated receptors. After washing and incubation with HRP-streptavidin, the membranes were visualized using a chemiluminescence-based detection method.
Cell viability assay
Tumor cells (2–3 × 103 cells/100 μL/well) in media supplemented with 10% FBS were plated in 96-well plates and cultured with the indicated compound for 72 h. After culturing, cell viability was measured using a CCK-8 kit (Dojindo Laboratories). The percentage of growth was determined relative to the untreated controls. Experiments were repeated at least three times with triplicate samples.
Patients and bioinformatics analysis
Paired colorectal tumor tissues and their corresponding adjacent nontumor colorectal tissues (5 cm away from the lesions) were collected from patients who underwent curative surgery for CRC at Renji Hospital, Shanghai, China. A CRC diagnosis was confirmed by histological examination, and the relevant clinical and pathological information was retrieved from the hospital database. The expression of microRNAs in CRC and normal controls was determined using the TCGA database, and a heat map and volcano map were acquired using R.
RNA extraction and quantitative real-time RT-PCR (qRT-PCR)
Total RNA was extracted from cell lines and frozen tumor samples using TRIzol reagent (Invitrogen, Carlsbad, CA). cDNA was reverse-transcribed from 1 µg of RNA using an SYBR® Prime ScriptTM RT-PCR kit (Takara Biochemicals, Tokyo, Japan), and quantitative PCR was performed using SYBR Select Master Mix (Roche, Switzerland) and gene-specific primers on an ABI PRISM® 7500HT Real-Time PCR System. The thermal cycling conditions were as follows: an initial step at 95°C for 15 s followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Each experiment was performed in a 20-µl reaction volume containing 10 µl of SYBR® Prime Ex TaqTM II (2×), 1 µl of forward primer and reverse primer (10 µM each), 2 µl of cDNA, and 7 µl of H2O. β-Actin was used as an internal control. The quantification of the mRNA was calculated using the comparative Ct (the threshold cycle) method according to the following formula: Ratio=2-∆∆CT= 2-[∆Ct(sample)-∆Ct(calibrator)], where ∆Ct is equal to the Ct of the target gene minus the Ct of the endogenous control gene (β-actin). The primers were as follows: EGFR (F: 5'-CCAAGGCACGAGTAACAAGC-3’; R: 5'- TCCCAAGGACCACCTCACAG-3’); ErbB3 (F: 5’-GGTGATGGGGAACCTTGAGAT-3’; R: 5’-CTGTCACTTCTCGAATCCACTG-3’)
EGFR T790M mutation detection
We purchased all probe qPCR mix (or UNG) reagents from Takara (Tokyo, Japan) and custom ordered primers and probes from GENEWIZ (Guangzhou, China). We performed probe qPCR analysis on an ABI PRISM®7500HT Real-Time PCR System as previously described under the following PCR conditions: EGFR T790M: forward primer, 5′-GCCTGCTGGGCATCTG-3′, reverse primer, 5′-TCTTTGTGTTCCCGGACATAGTC-3′. The probe sequences were 5′-VIC-ATGAGCTGCGTGATGAG-MGB-NFQ-3′ and 5′-FAM-ATGAGCTGCATGATGAG-MGB-NFQ-3′. Cycling conditions: 95°C × 10 min (1 cycle), 40 cycles of 94°C × 30 s and 58°C × 1 min, followed by a 10°C hold.
Antibodies and western blotting
Protein aliquots of 25 μg each were resolved by SDS polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA) with the appropriate antibodies. Electrophoresed protein samples were transferred to polyvinylidene difluoride membranes (Bio-Rad). After being washed three times, the membranes were incubated in blotting-grade blocker (Bio-Rad) for 1 h at room temperature and overnight at 4°C with primary antibodies to t-EGFR, p-EGFR, t-ErbB3, p-ErbB3, t-PI3K, p-PI3K, p-Akt, t-Akt, t-Erk1/2, p-Erk1/2, t-GSK3β,p-GSK3β, MMP9, PCNA,p21, Caspase-3, Cleaved-Caspase-3, Caspase-7, Cleaved-Caspase-7, Caspase-9, Cleaved-Caspase-9, and β-actin (1:1,000 dilution; Supplementary Table 1). After being washed three times, the membranes were incubated for 1 h at room temperature with an HRP-conjugated species-specific secondary antibody. Immunoreactive bands were visualized using SuperSignal West Dura Extended Duration Substrate Enhanced Chemiluminescent Substrate (Pierce Biotechnology). Each experiment was independently performed at least three times.
The RTCA xCELLigence system (ACEA Biosciences Inc., The Netherlands) was used to measure cell proliferation in real-time. CRC cells were placed at a density of 4000-8000/well, and E-plates were then transferred to the RTCA instrument for automated real-time monitoring under standard incubator conditions. Cell proliferation was monitored every 30 minutes. After 72 h, the measurement was stopped, and the results were analyzed using RTCA software and the results were analyzed after an additional 24 h.
Colony formation assay
Transfected CRC cells were seeded into 6-well plates at a density of 200 to 800 cells per well and incubated for 2 weeks. The cells were fixed and stained in a dye solution containing 0.1% crystal violet and 100% methanol. The number of colonies was subsequently counted and analyzed.
Apoptosis was determined by fluorescence-activated cell sorting (FACS) flow cytometry. The transfected cells collected after trypsinization were washed twice with PBS and resuspended in 1X binding buffer. Cells were then stained using 10 μL FITC-labeled buffer for 20 minutes and 10 μL PE-labeled buffer for 5 minutes according to the manufacturer’s instructions. The apoptosis rate was analyzed by FACS flow cytometry (BD Biosciences, Heidelberg, Germany).
Luciferase reporter assay
HCT116 cells grown in 24-well plates were co-transfected with luciferase reporter (200 ng per well), miR-323a-3p-mimic (200 ng per well), and 10 ng Renilla using Lipofectamine™ 2000. Forty-eight hours later, a Dual-Luciferase Reporter Assay kit (Promega, USA) was used to measure the luciferase and Renilla activities according to the manufacturer’s instructions. The relative luciferase activity was determined using BioTek Synergy 2 (BioTek, USA), and the transfection efficiency was normalized to Renilla activity.
Suspensions of 3 × 106 cells were injected subcutaneously into the flanks of four-week-old nude mice (Each group contained at least 5 mice). Once the mean tumor volume reached ~50–100 mm3, the mice were orally administered gefitinib and injected with agomiR three times per week. Tumors were measured three times per week using calipers, and tumor volumes were calculated as width2 × length/2. Mice were euthanized after 28 days, tumors were collected and weighed, and total RNA and protein were prepared from tumor tissues for qRT-PCR and western blot analysis.
All the data were obtained from experiments with adequate sample sizes and presented as the mean ± standard deviation (SD). Statistical analysis was performed using Prism 8 (GraphPad, San Diego, CA, USA). Two-way ANOVA, one-way ANOVA, χ2 tests, and the two-tailed unpaired Student’s t-tests were used to assess the significance of the differences between groups. The Kaplan–Meier method and log-rank tests of survival were used to study the role of miR-323a-3p. Each treatment was analyzed in at least three experiments.