The human CRC cell line HCT116 (3111C0001CCC000158, BMCR, China). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco Laboratories; Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco Laboratories), 100 units/mL penicillin and 100 µg/mL streptomycin (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA), and grown in a humidified atmosphere with 5% CO2 at 37˚C. Cells were cultured in a 75-cm2 flask or a 10-cm dish (Corning; New York, USA), with confluence maintained below 80%. After digestion with 0.25% (w/v) trypsin and 0.5 mM ethylenediaminetetraacetic acid (EDTA) (Gibco Laboratories), cells were passaged every 2-3 days with a subcultivation ratio of 1:3 to 1:8.
Plasmid constructs and siRNA oligos
The full-length cDNA for human METTL3 (GenBank accession No. NM_019852.5) was obtained from the cDNA library of HCT116 cells by reverse transcription polymerase chain reaction (RT-PCR). The vector used to generate full-length wild-type METTL3 and hsa_circ_0000523 was mammalian expression vector pcDNA3.1 (Thermo Fisher Scientific). The METTL3 coding sequence (CDS) or hsa_circ_0000523 fragment was subcloned into the pcDNA3.1 vector via KpnI and BamHI double-enzymatic sites. Positive clones were selected, and the plasmid expressing METTL3 or hsa_circ_0000523 was verified by sequencing. The siRNA oligos used in this study were designed and synthesized by Wuhan GeneCreate Biological Engineering Co., Ltd., Wuhan, China. The sequences for these siRNA oligos were as follows: siR-NC, sense 5’-GCUACUUCAGACGAGCAUUdTdT-3’, siR-METTL3, sense 5’-GCUGCACUUCAGACGAAUUdTdT-3’, siR-circ-NC; sense 5’- CAACAGAGCAAGAAGUAGAUdTdT-3’, and siR-circ-0000523, sense 5’- CAACAGAGCAAGAAGAUCUAdTdT-3’.
Transfection of plasmids and siRNA oligos
HCT116 cells were transfected with the empty pcDNA3.1 vector, METTL3-expressing pcDNA3.1 vector (pcDNA3.1-METTL3), negative control siRNA oligo (siR-NC), METTL3-specific siRNA oligo (siR-METTL3), negative control oligo for hsa_circ_0000523 (siR-circ-NC), or circ-0000523-specific siRNA oligo (siR-circ-0000523), using Lipofectamine 2000 reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions. HCT116 cells seeded in 6-well plates were maintained at confluence of 70% to 80% in complete culture medium. Then 2.5 μg plasmid or 100 pmol siRNA oligo was added to 250 μL Opti-MEM (Thermo Fisher Scientific), and 5 μL Lipofectamine 2000 reagent was added to 250 μL Opti-MEM. The diluted plasmid or siRNA oligo was mixed with diluted Lipofectamine 2000 reagent and kept at room temperature for 20 min. After 500 μL serum-free medium had been placed in each well, Opti-MEM mixture was added. After incubation of transfected cells at 37˚C for 4 h, the culture medium was changed to regular DMEM supplemented with 10% FBS.
RNA isolation and reverse transcription
Total RNA was extracted from cells with Trizol reagent (Thermo Fisher Scientific), according to the manufacturer's protocols. Briefly, after treatment, cell pellets were re-suspended in 1 mL Trizol and mixed well. Then, 0.2 mL of chloroform was added to the cell suspension, which was shaken vigorously for 15 s, then incubated for 3 min at room temperature. After centrifugation of the suspension at 12,000´g for 10 min at 4˚C, supernatant was collected and subjected to RNA precipitation by adding 0.5 mL of isopropanol at 4˚C. The RNA pellet was obtained by centrifugation at 12,000´g for 10 min at 4˚C, then washed with 75% ethanol. The appropriate amount of RNase-free distilled water was used to dissolve extracted RNA. The concentration of RNA was measured with a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific). For each sample, 1 μg of RNA was reverse-transcribed to cDNA with the ReverTra Ace qPCR RT Kit (catalogue number FSQ-101; Toyobo Co., Ltd.; Osaka, Japan), according to the specifications in the product manual.
Real-time quantitative PCR (RT-qPCR)
For RT-qPCR, SYBR Green Realtime PCR Master Mix (catalogue number QPK-212; Toyobo Co., Ltd.; Osaka, Japan) was used to measure the expression of targeted genes, according to the manufacturer’s instructions. For quantification of hsa_circ_0000523 and let-7b, U6 was used as an internal reference gene. GAPDH was used as an internal reference gene for quantification of METTL3. The conditions for PCR were set as follows: pre-denaturation at 95°C for 1 min, followed by 40 cycles of denaturation at 95°C for 15 s, and annealing and elongation at 60°C for 30 s. The 7900HT Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific) was used to perform the assay. The 2-ΔΔCt method was used to calculate differences between the experimental and control groups in expression of the target gene. The calculation formula was as follows: ΔΔCt = ΔCt experimental group - ΔCt normal group, ΔCt = Ct target gene – Ct internal reference. The sequences of primers used for RT-qPCR are presented in Table 1.
CCK-8 assay to measure cell proliferation
After transfection of empty pcDNA3.1 vector, METTL3-expressing pcDNA3.1 vector, siR-NC oligo, or siR-METTL3 oligo, HCT116 cells were seeded into flat-bottomed 96-well plates at a concentration of 3×103–6×103 cells per well, in 100 μL culture medium. Each treatment condition was replicated in 9 wells. Cells were incubated for 24 h, 48 h or 72 h in an incubator with 5% CO2 at 37˚C. Cell proliferation was assayed with the CCK-8 assay (Beyotime Biotech Inc, Shanghai, China), according to the manufacturer’s instructions. Briefly, 20 μL of 5 mg/mL CCK-8 reagent was added to the culture medium, and cells were further cultured for an additional 4 h. The optical density (OD) values of all wells were measured with a plate reader (Thermo Fisher Scientific) at 450 nm.
HCT116 cells seeded in 6-well plates were transfected with empty pcDNA3.1 vector, METTL3-expressing pcDNA3.1 vector, siR-NC oligo, or siR-METTL3 oligo. At 48 h after transfection, cells were detached by incubation with 0.25% trypsin-EDTA solution (Gibco Laboratories), then harvested. Flow cytometric analysis was performed to detect apoptosis using the Annexin V-fluorescein isothiocyanate (FITC) /propidium iodide (PI) Apoptosis Kit (Beyotime Biotech Inc., Shanghai, China), according to the manufacturer’s protocol. Briefly, after one wash in phosphate-buffered saline and one wash in binding buffer, cells were stained with Annexin V-FITC/PI for 20 min at room temperature, in the dark. After another wash in binding buffer, labeled cells were detected immediately by a flow cytometer (CytoFLEX S, Beckman Coulter; Brea, CA, USA). Data were analyzed with CytExpert Software (Beckman Coulter).
The cellular potential for migration was determined using a 24-well transwell plate with 8.0-μm Pore Polyester Membrane Inserts (Corning; Thermo Fisher Scientific). Matrigel (Corning) was melted overnight at 4˚C and diluted to a final concentration of 1 mg/mL in pre-cooled serum-free medium. Then, 100 mL of diluted matrigel was added to the bottom of the upper chamber. The plate was then incubated at 37˚C for 4-5 h to dry the matrigel. At 24 h after transfection, HCT116 cells in the logarithmic growth phase were seeded in triplicate at a density of 1×106 cells/well. Cells were seeded on top of the transwell plate, in 100 μL DMEM supplemented with 0.1% bovine serum albumin. Then 0.8 mL of DMEM supplemented with 10% FBS was added to the lower chamber as a chemoattractant. After 24 h of incubation, cells on the top surface of the insert were removed with a cotton swab. Cells that had migrated to the lower surface of the membrane were fixed with 4% paraformaldehyde, stained with 800 μL Giemsa solution (Beyotime Biotech Inc., Shanghai, China). Cells were visualized by a microscope (CKX-41; Olympus, Japan). Counts were obtained for three randomly selected optical fields.
All data were analyzed using SPSS Version 21.0 software (IBM Corp., USA). These data are presented as mean ± standard deviation (SD). Quantitative data were compared using the chi-square test. Comparisons between groups were performed with Student's t-test. P < 0.05 was accepted as an indication of statistical significance.