Clinical tissue specimens
Thirty CRC, thirty adenoma and thirty adjacent normal tissues (normal) were obtained during surgery in the Longhua Hospital affiliated with Shanghai University of Traditional Chinese Medicine. The diagnosis of CRC and adenoma was confirmed based on pathological evidence. Tissues were snap-frozen in liquid nitrogen and stored at -80°C before detection. The study was approved by the Ethics Committee of Longhua Hospital (2019LCSY020), and informed consent was obtained from all participants.
RNA m6A quantification assay
M6A level was assayed using an RNA m6A quantification kit (ab185912, abcam, USA) according to previous study . In brief, 200ng RNA was incubated for 60 min with the capture antibody; and then the detection antibody and enhancer solution were added. Finally, samples were incubated with developer solution for 10 min. The absorbance was detected at a wavelength of 450 nm.
Tissue samples from the normal, adenoma, and CRC groups were fixed, and then cut into 4-µm sections for immunohistochemistry (IHC). Tissue microarrays (TMAs) were obtained from Shanghai Outdo Biotech Co., Ltd (Shanghai, China), and IHC was performed. In brief, samples were incubated with the m6A antibody (56593, CST, USA), and METTL3 antibody (ab195352, Abcam, USA) overnight at 4◦C. Subsequently, the secondary antibodies were incubated for 1 h at 37◦C. Finally, samples were stained, and then imaged. The scores of IHC were performed.
The m6A antibody (56593, CST, USA), METTL3 antibody (ab195352, Abcam, USA), and CRB3 antibody (PA5-53092, Thermo Fisher, USA) were obtained. Samples were incubated with the m6A antibody, METTL3 antibody, and CRB3 antibody overnight at 4◦C. Subsequently, the secondary antibodies conjugated with Alexa Fluor were incubated for 1 h at 37◦C. Finally, samples were stained, and then imaged.
Cell culture and transfection
Normal colon cells (FHC) and CRC cells (HCT116, HT29, SW480 and SW620) (Shanghai Cell Bank, shanghai, China) were cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin (100 U/mL) in an incubator with 5% CO2 at 37°C. In addition, 293T cells obtained from ATCC were cultured in RPMI Medium 1640 with 10% fetal bovine serum and penicillin/streptomycin (100 U/mL) (Gibco, Carlsbad, USA). METTL3 short hairpin RNA plasmid (sh-METTL3), CRB3 short hairpin RNA plasmid (sh-CRB3) or negative control (sh-NC) (Genomeditech, China) were transfected using FuGene® HD transfection reagent (Promega, USA) according to a previous study .
After transfection, HCT116 and SW620 cells were seeded into 96-well plates at a concentration of 1×104 cells and cultured for 0, 24, 48 and 72 h. Then, 10 µl CCK8 was added to each well. After incubation at 37°C for 1 h, the absorbance value was detected at 450 nm.
Wound healing assay
After transfection, HCT116 and SW620 cells were seeded in a six-well dish with a culture insert (Ibidi, Germany) at a concentration of 3×104 cells. After 24 h, the culture insert was removed, and the cells were washed twice with phosphate buffer. Then 2ml serum-free medium was added to each dish for 48 h. Images were captured, and the wound area was measured using ImageJ software (National Institutes of Health, USA).
Six-well plates with 8-µm chambers (Corning, USA) were used to assess cellular migration (without Matrigel) or invasion (with Matrigel). Briefly, transfected HCT116 and SW620 cells were seeded in 6-well plates at a concentration of 1×105 cells. 200µl serum-free medium was added to the upper chamber, and 600 µl of medium with 30% fetal bovine serum was added to the lower chamber for 48 h. Then, the cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet solution for 15 min. Five fields were randomly selected to calculate the number of migrating or invading cells.
Quantitative real-time PCR
Total RNA was extracted using TRIzol reagent (Ambion, USA). cDNA was synthesized using an EVM-MLV reverse transcription kit (Aikeri Biotech, Hunan, China). The amplification reaction was performed using the SYBR-Green qPCR kit (Thermo Fisher Scientific, MA, USA). Gene expression was normalized using β-actin. The primers were listed in Additional file 3 Table S1.
Cells were collected and lysed. Protein concentration was determined. The protein was separated and transferred to a PVDF membrane followed by incubation with 5% milk at room temperature for 1 h. The membrane was incubated at 4°C overnight with the following antibodies as indicated: METTL3 (86132, CST, USA), CRB3 (NBP1-98328, Novus Biologicals, USA), YTHDF2 (80014, CST, USA), MST1 (3682, CST, USA), phospho-MST1 (49332, CST, USA), SAV1 (13301, CST, USA), LATS1 (3477, CST, USA), phospho-LATS1 (8654, CST, USA), MOB1 (13730, CST, USA), phospho-MOB1 (8699, CST, USA), YAP (4912, CST, USA), phospho-YAP (13008, CST, USA), Histone (4499S, CST, USA), and β-actin (4970s, CST, USA). Then, the secondary antibody was added and incubated at room temperature for 1 h, and protein expression was observed using a chemiluminescence gel imaging system (Tanon 5200, China).
Human m6A Epitranscriptomic microarray analysis
Total RNA was quantified using the NanoDrop ND-1000. The sample preparation and microarray hybridization were performed based on the Arraystar’s standard protocols. Briefly, the total RNAs were immunoprecipitated with anti- m6A antibody. The modified RNAs were eluted from the immunoprecipitated magnetic beads as the “IP”. The unmodified RNAs were recovered from the supernatant as “Sup”. The “IP” and “Sup” RNAs were labeled with Cy5 and Cy3 respectively using Arraystar Super RNA Labeling Kit. The RNAs were combined together and hybridized onto Arraystar Human m6A Epitranscriptomic Microarray (8x60K, Arraystar). After washing the slides, the arrays were scanned by an Agilent Scanner G2505C.
Data Processing and Analysis
Agilent Feature Extraction software was used to analyze acquired array images. Raw intensities of IP (Cy5-labelled) and Sup (Cy3-labelled) were normalized. After normalization, the probe signals were retained for further “m6A methylation level” and “m6A quantity” analysis. “m6A methylation level” was calculated for the percentage of modification based on the IP (Cy5-labelled) and Sup (Cy3-labelled) normalized intensities. “m6A quantity” was calculated for the m6A methylation amount based on the IP (Cy5-labelled) normalized intensities. Differentially m6A-methylated RNAs between two comparison groups were identified by filtering with the thresholds of fold change (FC) > 1.5 and P value < 0.05.
RNA stability assay
HCT116 cells were seeded in 6-well plates for 24h, and then treated with 5 µg/mL actinomycin D (MCE, USA) at the 0, 2, 4, 8, 24 h. Total RNA was then isolated by TRIzol (Ambion, USA) and analyzed by qPCR. The mRNA expression for each group at the indicated time was calculated and normalized by β-Actin.
Double luciferase reporter assay
Here, 293T cells were seeded in 24-well plates and transfected using FuGene® HD transfection reagent (Promega, USA) according to a previous study . Briefly, 293T cells were transfected with CRB3 wild type (CRB3-WT; Genomeditech, China) or CRB3 mutant (CRB3-Mut; Genomeditech, China) plasmid with or without METTL3 knockdown for 6 h. Luciferase activity was detected using the dual-luciferase reporter system (Promega, USA).
Statistical analysis was conducted using SPSS 24.0 software. Data were assessed using a two-tailed Student’s t-test. Survival curves were generated using the Kaplan–Meier method and compared using the log-rank test. Survival data were performed by univariate and multivariate Cox regression analyses. The distribution differences of the variables were analyzed by the Pearson’s chi-square test. P < 0.05 was considered statistically significant.