Clinical CRC samples
Ethics approval was obtained from the Human Research Ethics Committee of the Second Affiliated Hospital School of Medicine, Zhejiang University (Approval number: 20220034). For m1A MeRIP sequencing, three pairs (tumor and adjacent non-tumorous tissues) of clinical samples were obtained from patients with CRC in the Second Affiliated Hospital, School of Medicine, Zhejiang University with written consent forms. All tissues were stored at -80°C before use. For immunohistochemistry (IHC), 177 pairs of paraffin-embedded tissues, including three cohorts (cohort 1:90 pairs; cohort 2:30 pairs; and cohort 3:57 pairs), were collected from the archive cabinet of the pathology department. Accordingly, three tissue arrays were prepared in cooperation with Outdo Biotechnology (Shanghai, China).
IHC
Following deparaffinization, rehydration, and antigen retrieval, tissue sections were conjugated with DIAPH3 antibody at 4°C overnight and then incubated with HRP-conjugated secondary antibody for 30 min. After washing the slides three times with PBS, the color was developed using the DAB Chromogen. The slides were then rinsed with tap water and counterstained with hematoxylin. Five random fields per section were viewed under a light microscope (Leika, Wetzler, Germany), and the expression of DIAPH3 was scored in terms of the intensity of staining and the percentage of positively stained cells. The proportion of positive cells in each specimen was quantified under a microscope and the cells were classified into four groups. 0:0–5% positive cells; 1:6–50% positive cells; 2:51–75% positive cells; 3:76–100% positive cells. The intensity of DIAPH3 staining was graded as follows: 0, no staining; 1, weak staining; 2, moderate staining; 3, dense staining (Supplementary Fig. S1). Multiplying the score of the intensity by the proportion of positive staining was defined as the DIAPH3 staining score.
Cell culture and transfection
Two human CRC cell lines, HCT116 and RKO, were purchased from the American Type Culture Collection and cultured in McCoy's 5A and EMEM medium, respectively. The medium was supplemented with 10% fetal bovine serum (FBS; ExCell Bio, Taicang, China) at a 37°C atmosphere of 5% CO2. Cells were plated in a 6-well dish 18 h before transfection. Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was used to deliver plasmids into CRC cells according to the manufacturer’s instructions. siRNAs targeting human DIAPH3 (Tsingke Biotechnology Company, Beijing, China) were transfected with Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA, USA) in Opti-MEM (Gibco, California, USA). The siRNAs sequences were: si-DIAPH3-1, sense 5'-GAGUGAAUAUAGCAACUUATT-3′; si-DIAPH3-2, sense 5′-GAGUGAAUAUAGCAACUUATT-3′; si-TRMT6, sense 5'-AGAUCGAAGUCAUCCUAAACU-3′.
Mining the database
We used the GEPIA2 website (http://gepia2.cancer-pku.cn/) to analyze DIAPH3 expression in colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ). Correlation analysis between DIAPH3 and m1A regulators was performed using GEPIA. Survival analysis of DIAPH3 was performed using the online database GenomicScape (http://www.genomicscape.com/) [14]. The sample information and expression data are available in the Gene Expression Omnibus (GEO) database [Accession nos. GSE17538].
RNA Extraction and Quantitative Real-Time PCR (RT-qPCR)
Total RNA was extracted from tissue samples and cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. One microgram of RNA per sample was reverse-transcribed to cDNA using HiScript II Q RT SuperMix for qPCR (Vazyme, Nanjing, China), and the latter was amplified using ChamQ Universal SYBR qPCR Master Mix (Vazyme). The relative expression of DIAPH3 was calculated by the 2-∆∆Ct method, with GAPDH as the internal control. The primer sequences are listed in Table S1.
MeRIP-seq and MeRIP-qPCR
Total RNA was isolated from the tumor and adjacent normal tissues using Trizol (Invitrogen, Carlsbad, CA, USA) and sent to Cloud-Seq Biotech for m1A RNA immunoprecipitation (RIP) sequencing. RNA quality assessment, library construction, and rough handling of raw data were performed as previously described [11]. Peaks with p value < 0.05 and q value < 0.1 were screened out. To screen for genes that were not rare, transcripts with FPKM > 10 in either CRC or adjacent normal tissues were selected. If more than one m1A peak was acquired for a single transcript, only the peak that changed the most was selected for further analysis.
For MeRIP-qPCR, One-tenth of the total RNAs was reserved as input. The remaining RNA was immunoprecipitated with m1A antibody in RIP buffer (10 mM Tris HCl, 150 mM NaCl, 0.1% NP-40, and 0.2% RNase inhibitor) supplemented with RNase inhibitors for 2 h at 4°C. Above mixtures were then added into Protein A/G beads (Invitrogen, California, USA), prewashed with RIP buffer, and rotated for an additional 2 h at 4°C. After washing the sediment five times with RIP buffer, we eluted RNA from the beads. Then glycogen and isopropanol were added into the supernatant and incubated for 4 h at 4°C. After the eluted RNA was washed with ethanol, the pellet was resuspended in RNase-free water for further use. Finally, the eluted and input RNAs were reverse-transcribed and analyzed using qPCR. The relative enrichment of m1A containing mRNA was calculated by normalization to the input.
Western Blotting
Cells were lysed using radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China) supplemented with phenylmethanesulfonyl fluoride (Beyotime). Proteins were quantified using a BCA Protein Assay Kit (Fude Biological Technology, Hangzhou, China) and equal amounts per sample were separated by SDS-PAGE and transferred onto a PVDF membrane (Millipore). The membranes were blocked with 5% skim milk for 1 h at at 25°C, and then incubated overnight with primary antibodies against DIAPH3 (Proteintech, Wuhan, China), TRMT6 (Abclonal, Wuhan, China), BRD9 (Abclonal), keratin 19(KRT19, Abclonal), SMYD3 (Abclonal), ARL2 (Abclonal), RIN1 (Abclonal), CAVIN3 (Abclonal), GGNBP2 (Absin, Shanghai, China), and GAPDH (Diagbio, Hangzhou, China) at 4°C. After incubation for 1 h with an HRP-conjugated secondary antibody (Beyotime, Shanghai, China) at 25°C, the membranes were washed three times with TBST, and the bands were visualized using an enhanced chemiluminescence reagent (NCM Biotech, Suzhou, China).
Scratch wound assay
The CRC cells were cultured in 24-well plates. After overnight incubation, we scratched the area in the middle of the well with a 10 µL pipette tip and washed the floating cells with PBS. At 0 and 24 h after wounding, images were captured using a light microscope (Leika, Wetzler, Germany). The width between the two boundary lines was measured using IMAGEJ software version 1.8.0.345 (NIH, Maryland, USA). Percentage of relative closure (%) = (width on day 0 - width on day 24)/width on day 0 × 100.
Cell Migration and Invasion Assays
The cell migration and invasion assays were performed using 24-well Transwell apparatus (8-µm pore size, Corning, NY, USA). A total of 8 × 104 cells per well were seeded in the uncoated upper chamber for cell migration assays, whereas 10 × 104 cells were seeded in Matrigel (Nova Pharmaceutical Technology, Shanghai, China)-coated chambers for cell invasion assays. The lower chambers were filled with a medium containing 20% FBS. After 48 h of incubation at 37°C, the cells that had migrated from the upper to the lower surface of the filters were washed thrice with PBS, fixed in 4% paraformaldehyde for 15 min, stained with 0.05% crystal violet for 10 min at 25°C, and washed twice with PBS. The cells were counted in five random fields (200× magnification) per sample under a light microscope (Leika, Wetzler, Germany).
CRISPR/Cas9-driven gene knockout
The genomic deletion of DIAPH3 in colon cell lines (HCT116 and RKO) was carried out using CRISPR/Cas9 vector with a specific DIAPH3 guide RNA sequence (gRNA:5′- CTCTGCGGTATGCATTGTAG-3′). After 48 h of transfection, single cells were sorted by FACS based on GFP expression in 96-well plates to obtain DIAPH3 knockout (KO) clones. Complete KO was confirmed by Sanger sequencing and western blotting using a DIAPH3 antibody.
Mass Spectrometry (MS) analysis
After digesting the protein suspension overnight at 37°C with trypsin (Beyotime, Shanghai, China), TMT reagent (Invitrogen, California, USA) was added to label the resulting peptides. The complexes were sent to LC Sciences (Hangzhou, China) for MS analysis, and reversed-phase chromatography was conducted to fractionate the TMT-labeled peptides. The fractionated peptide mixtures were loaded onto a C18-reversed phase analytical column and separated by mixing formic acid with acetonitrile. LC–MS/MS experiments were performed in positive ion mode on a Q Exactive Plus mass spectrometer (Invitrogen, California, USA). The MS data were obtained by selecting the most abundant precursor ions from the survey scans. The output files were processed using the MASCOT engine (Matrix Science, version 2.6) embedded in Proteome Discoverer 2.2. The tolerance for the precursor mass was 10 ppm, and that for the fragments was 0.05 Da. Proteins with a fold change > 1.3 and a p value < 0.1 were identified as differentially expressed proteins.
Xenograft metastatic model
All animal experiments were approved by the Animal Care and Use Committee of the Second Affiliated Hospital School of Medicine, Zhejiang University (Approval number: 2022002). Female BALB/c nude mice (aged 4 weeks) were purchased from the Institute for Experimental Animals of the Chinese Academy of Vital Rivers (Beijing, China). All the mice were housed in a specific pathogen-free environment at the Animal Facility of the Second Affiliated Hospital of Zhejiang University School of Medicine. After the DIAPH3 KO clone was obtained, cells were transfected with PGMLV-CMV-luc-PGK-neo. We injected 2 × 106 DIAPH3-KO or Negative-KO HCT116 cells into mice intraperitoneally (n = 5). After 28 days, the mice were intraperitoneally administered D-luciferin (1.5 mg) and imaged weekly using an IVIS (PerkinElmer IVIS Lumina XRMS Series III imaging system) to measure the luciferase signal intensity. Once a significant difference in luciferase signal intensity was observed between the two groups, the mice were sacrificed.
Statistical Analysis
Each experiment was repeated at least thrice. Two groups were compared using a two-tailed unpaired Student’s t test. Chi-square tests were used to analyze the significance of categorical data. Survival rates were calculated using the Kaplan–Meier method and compared using the log-rank test. Statistical analyses were performed using GraphPad Prism 8 software. Unless otherwise specified, p < 0.05 was considered statistically significant.