2.1 TCGA databases and associated analysis tools
TCGA- Colon Adenocarcinoma (TCGA-COAD, https://cancergenome.nih.gov/) contains 480 colon cancer cases and 41 normal control cases, includes basic information such as age, sex, race, history, type of diagnosis, tumor grade stage. The expression of genes was analyzed and visualized by UALCAN website tool (http://ualcan.path.uab.edu/). The overall survival analysis was performed by GEPIA website tool (http://gepia.cancer-pku.cn/). The co-expression of m6A regulated enzymes was analyzed and visualized by R software based on Pearson Correlation Coefficient analysis. Gene ontology (GO) and KEGG pathway analysis was analyzed by Database for Annotation Visualization and Integrated Discovery (DAVID, david.ncifcrf.gov/) online tool and visualized by R software. Somatic mutation files were summarized, analyzed and visualized through the R software package “maftools”. Copy number data were analyzed and visualized through the R software package “RCircos”.
2.2 Patients and specimens
Colon cancer specimens and paired non-tumor bowel tissues were collected from July 2017 to July 2019. Patients with the following criteria were excluded from participation: had received adjuvant chemotherapy or radiotherapy prior to surgery; had additional cancers diagnoses. All patients were classified according to the 7th edition of the TNM staging system 23. Postoperative adjuvant therapies were performed, according to standard schedules and doses. All participating patients gave their written informed consent. This study was approved by the Ethical Committee of Shanghai Pudong Hospital.
2.3 Immunohistochemical (IHC) staining
IHC was performed on paraffin-embedded sections. The sections were deparaffinized in xylene and hydrated with decreasing concentrations of ethanol (100, 90, 80, 75%) for 3 min each time and microwaved-heated in sodium citrate buffer for antigen retrieval. Then, the sections were blocked in 5% BSA and incubated with anti-IGF2BP3, VEGF, CD31, Ki67 rabbit polyclonal antibody (1:200; ProteinTech Group, Inc., Wuhan, China) at 4°C overnight. Next, the sections were treated with horseradish peroxidase (HRP)‑conjugated rabbit secondary antibody (1:200; ProteinTech Group, Inc.) for 60 min at room temperature; then, 3,3’‑diaminobenzidine development (DAB Substrate Chromogen System; Dako, Denmark) and hematoxylin staining were performed. The sections were fixed and images were obtained with inverted microscope (Olympus IX71, Japan).
2.4 Western Blotting Analysis
The total cellular proteins from each group were extracted using RIPA lysis buffer with 1% phenylmethanesulfonyl fluoride (PMSF). Then, equal amounts (20 μg) of protein determined by BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) were separated using 10% SDS-PAGE gels. The proteins were then transferred to PVDF membranes (0.45 mm, Solarbio, Beijing, China). The membranes were blocked with 5% nonfat milk for 1 h at room temperature and then incubated with anti-IGF2BP3, Cyclin D1 (1:1000, Proteintech Group. Inc) rabbit polyclonal antibodies at 4oC for 12 h. anti-β-actin rabbit polyclonal antibody (1:4000, Proteintech Group. Inc) was used as loading controls and normalization. The secondary antibodies were anti-mouse or anti-rabbit antibody and conjugated to horseradish peroxidase (HRP) (1:4000, Proteintech Group. Inc). The secondary antibodies were used at a 1:4000 dilution and were incubated for approximately 1 h at room temperature. The bands were visualized with ECL reagents (Thermo Fisher Scientific) and developed by Omega Lum G (Aplegen, USA).
2.5 Cell Culture and knockdown of IGF2BP3
Human colon cancer cell lines HCT116, RKO, SW480, SW620, SW1116, LoVo and HT29 were purchased from the University of Colorado Cancer Center Cell Bank. The cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA) at 37°C in a 5% CO2 atmosphere. Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from Allcells, Inc. (Alameda, CA, USA) and cultured in Endothelial Cell Medium (ECM; ScienCell Research Laboratories, Carlsbad, CA, USA) supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA).
The shRNAs of human IGF2BP3 (sequence: GCTGCACTTCAGACGAATTAT) was synthesized by Genomeditech,lnc. (Shanghai, China) and cloned into the pCDH-CMV-MCS-EF1-Puro lentiviral vector to construct the pCDH-shIGF2BP3 knockdown plasmids. In accordance with the instructions of the product manual, Lipofectamine 3000 (Invitrogen, Inc.) was used to co-transfect the target plasmid or the scrambled vector, psPAX2, PMG.2G into the HEK293T tool cells to obtain IGF2BP3 knockdown lentivirus or scrambled control lentivirus. Then, the lentivirus (multiplicity of infection, MOI=10) was used to infect HCT116 and RKO. The IGF2BP3 knockdown cell lines HCT-sh1, sh2/RKO-sh1, sh2 and negative control cell lines HCT-scr/RKO-scr was screened by puromycin (2μg/mL, 72h). The knockdown of IGF2BP3 was confirmed by Western blotting.
2.6 RNA extraction, reverse transcription and quantitative PCR (RT-qPCR)
Total RNA was extracted by Trizol Regent (Invitrogen) from cells. cDNA was obtained from total RNA with PrimeScript™ RT reagent kit (Takara Bio, Inc., Otsu, Japan). The mRNA expression was assessed by Real‑time quantitative PCR, which was carried out in triplicate by a SYBR Premix Ex Taq™ kit (Takara Bio) and ABI 7900HT Real‑Time PCR system (Applied Biosystems Life Technologies, Foster City, CA, USA). The primers for RT-qPCR were showed in Table 1. The comparative cycle threshold values (2‑ΔΔCt) were adopted to analyze the final results.
2.7 Cell Cycle assay
For cell cycle assay, 1x106 cells were harvested, fixed in 70% ethanol, and stored at 4˚C overnight. Cells were then stained with PI staining solution for 30 min in the dark at room temperature followed by flow cytometry. The fractions of the cells in G1, S, and G2/phases were calculated with Modfit software (Verity Software House, USA).
2.8 RNA immunoprecipitation (RIP)
For RIP assay, cells were irradiated twice with 400 mJ/cm2 at 254 nm by Stratalinker on ice and lysed with RIP lysis buffer (300 mM NaCl, 0.2% NP-40, 20 mM Tris-HCl PH 7.6, 0.5 mM DTT, protease inhibitor and RNase inhibitor) at 4 °C through disruptive sonication. Then the lysis was incubated with 5μg anti-IGF2BP3 Rabbit antibody, or IgG (ProteinTech Group) pre-conjugated protein A/G Magnetic Beads (Millipore) in 500 μl IP buffer (150 mM NaCl, 10 mM Tris–HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 1 % Triton X-100, 0.5 % NP-40) supplemented with RNase inhibitors (Thermo Fisher) at 4 °C overnight. The IP complex was treated with Proteinase K (Thermo Fisher) for 1h at 52 °C, and RNA was purified with phenol:chloroform:isoamyl alcohol. Finally, RT-qPCR was performed as described above. The primers for RT-qPCR were showed in Table 1.
Intact total RNA was extracted via centrifugation column (MiniBEST Universal RNA Extraction Kit; Takara) and mRNA was further purified via polyATtract mRNA Isolation Systems (Promega Corp.). Subsequently, m6A RNA immunoprecipitation (MeRIP) was performed with Magna MeRIP m6A kit (17-10499, Millipore) according to the manufacturer’s instructions. The IP production was performed RT-qPCR as described above. The primers for RT-qPCR were showed in Table 1.
2.10 RNA stability assay
The cells were treated with Actinomycin D (MedChemExpress) at 5 μg/ml. After incubation for 0h, 2h and 4h, the cells were collected and RNA was extracted for RT-qPCR as described above. The mRNA degradation rate was estimated according to published protocols. The degradation rate of RNA (K) was estimated by following equation:
where t is the transcription inhibition time, and Nt and N0 are the RNA quantities at time t and time 0. The RNA lifetime (t1/2) can be calculated from the degradation rate as follows:
2.11 Clone formation test
For this assay, 500 cells were seeded into 6-well plates and incubated at 37℃. Clone size was observed daily under a microscope until the number of cells in the majority of clones was >50. Then, the medium was removed and the cells were stained with 0.2% crystal violet for 30 min. The cells were washed 3 times with PBS, then photographed and the clones were counted. The ratio of clone formation was calculated with the following equation: Ratio of clone formation (%) = clone number / 500 x 100.
2.12 Cell proliferation assay
3×103 cells suspended in 100ul RPMI-1640 medium were seeded into 96-well plate. The cell proliferation was assessed by the CCK8 (Dojindo Molecular Technologies, Japan). 10ul CCK8 solution was given to each well of the plate after different incubation times: 0h, 24h, 48h and 72h. Finally, we measured the absorbance at 450nm wavelength after 2h incubation.
2.13 DNA replication analyzed by EdU Assay
Cell proliferation was measured by 5-ethynyl-2′-deoxyuridine (EdU) assay using an EdU assay kit (UE Everbright, Inc.) according to the manufacturer’s instructions. Brieﬂy, 104 cells suspended in 500ul RPMI-1640 medium were seeded per well in 24-well plates and cultured for 48 h. The cells were then exposed to 50 µM of EdU for additional 2 h at 37°C.Than the cells were fixed with 4% formaldehyde for 15 min at room temperature and treated with 0.5% Triton X-100 for 20 min at room temperature for permeabilization. After 3× washes with PBS, the cells were treated with 200 µL of reaction buffer for 30 min. Subsequently, the DNA contents of each well of cells were stained with 200 µL of Hoechst 33342 (5 µg/mL) for 30 min and visualized under a ﬂuorescent microscope.
2.14 Measurement of VEGF via ELISA assay
The concentration of VEGF in cell culture medium was measured by a commercial ELISA kit (R&D Systems). In brief, a specific anti-VEGF monoclonal antibody was coated onto a microplate. Standards and samples were added to microplate. VEGF was detected with biotinylated goat anti-VEGF antibody and peroxidase-conjugated streptavidin. Peroxidase substrate was added and the reaction stopped using Stop solution. Absorbance was measured at 450 nm and absolute protein levels were interpolated from the standard curve.
2.15 Construction of HCT116 derived conditioned medium
HCT-116-scr, HCT-sh1, HCT-sh2 were incubated in serum-free medium for 24 hours, after which culture supernatants were collected as conditioned medium (CM). CM was centrifuged at 3,000 rpm to remove debris and then stored at −80°C. In all HUVECs related assays, CM was substitute for ECM medium.
2.16 Cell invasion assays
Cell invasion were analyzed with transwell plates (24-well insert, 8 μm pore size; BD Biosciences, Bedford, MA, USA). The filters (Corning Inc., USA) were coated with 55 μL Matrigel (1:8 dilution; BD Biosciences). The 104 HUVECs were suspended in 100μl CM without serum and seeded in the upper chamber. Next, 600μl 90% CM supplement with 10% FBS was added to the bottom chamber. After incubation for 24h, the chambers were fixed by 4% paraformaldehyde for 30 min and then stained by 0.1% crystal violet for 30 minutes. At last, we used a magnification microscope to count the amount of the invasion cells in the bottom of the chamber.
2. 17 Tube formation assay
100 μL Matrigel (BD Biosciences) was planted into precooled 96-well plates on ice and incubated for 30 min at 37 °C. Then, HUVECs cells pre-cultured with CM (supplemented with 10% FBS) for 18h was harvested and suspended in 100 μl CM. HUVECs were seeded to the wells with incubating at 37 °C for another 6 hours. Finally, the HUVECs cells were stained by calcein-AM ((Invitrogen, Inc.) and captured with a fluorescence microscope (Nikon, Tokyo, Japan). The number and length of tubes were counted and analyzed by ImageJ (Version 1.8.0, National Institutes of Health).
2.18 Subcutaneous xenografts of nude mice
5-week-old male Balb/c-nu mice were provided by the Beijing Vital River Laboratory Animal Technology Co. Ltd. All detailed experimental procedures were approved by the Institutional Animal Care and Utilization Committee of Fudan University Pudong Animal Experimental Center. All the mice (n = 12) were equally and randomly divided into the HCT-scr and HCT-shMETTL3 group. 3×106 HCT-scr or HCT-shIGF2BP3 cells suspended in 100µl PBS were injected subcutaneously from the axilla of each nude mice. After 1 weeks, the long (L) and short (S) diameter of the tumors were measured with vernier caliper every 3 days (tumor volume=L*S2/2). The growth curve of subcutaneous tumors was drawn on the basis of the measured tumor volume. All mice were killed after 17 days since injection of colon cancer cells and subcutaneous tumors were removed completely. The tumors were weighed and performed into paraffin section. The evaluation of vascular density in xenografts was analyzed as microvascular density (MVD). The vessels were labeled by IHC of CD31. The area of densest plaque neovascularization (hot plot) was identified in each plaque under in the low power lens (magnification: 100x). Subsequently, at least 5 high-power fields in the hot plot were captured (magnification: 400x). Any single cell or cell mass stained with CD31, as long as it has a clear separation from the surrounding cells, it is considered to be a countable microvascular. The average microvascular number of at least 5 fields was identified as the MVD of the section.
2.19 RNA m6A quantification
Total RNA was extracted via TRIzol (Invitrogen, CA, USA) as described below, and RNA quality was assessed by NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA). The m6A modification level of total RNA was examined via EpiQuik m6A RNA Methylation Quantification Kit (p-9005; Epigentek Group Inc., Farmingdale, NY, USA) according to the instruction. Briefly, 200 ng RNA accompanied with m6A standard were coated on assay wells, followed by capture antibody solution and detection antibody solution. The m6A levels were quantified colorimetrically by reading the absorbance of each well at a wavelength of 450 nm (OD450), and then calculations were performed based on the standard curve.
2.20 Luciferase reporter assay
The wild type (VEGF-wt) and m6A sites mutated VEGF (VEGF-mut) were constructed into luciferase reporter vector pGL3-Rluc and followed by Dual-Glo Luciferase Assay system ((Promega Corp., Madison, WI, USA). After 36h transfection, the cells were lysed by passive lysis buffer. Firefly Luciferase (F-luc) and Renilla Luciferase (R-luc) of lysis were detected respectively. 2.21 Statistical analysis
All the experiments were performed 3 times at least. SPSS software (version 19.0, IBM Corp., Armonk, NY, USA) was used for statistical analysis of all the experimental data. GraphPad Prism (version 7, GraphPad Software, La Jolla, CA, USA) was used to determine the statistical results. All data are expressed as the mean + standard deviation (mean + sd). The statistical analysis of the data from 2 groups was performed using a t-test. The comparisons of multiple groups were performed by one-way ANOVA and then an LSD-t test. P <0.05 was considered to be significant.