Tissue samples and cell lines
Sixty-three paraffin-embedded human CRC tissues and corresponding normal mucosal tissues and sixteen fresh human CRC tissues and matched normal mucosal tissues were collected for this study. All the samples were obtained from the Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou, China (from March 2018 to June 2019). All samples were obtained from patients who had been diagnosed with primary CRC by two pathologists based on microscopic analysis of tumor tissue, and none of the patients received radiotherapy or chemotherapy prior to surgical resection. Sixty-three cases, including 38 (60.3%) males and 25 (39.7%) females, were recruited for this study. The use of human materials was approved by the Medical Ethics Committee of Nanfang Hospital, Southern Medical University. Seven human CRC cell lines (HCT8, SW620, Caco2, HT29, HCT15, HCT116, and LOVO) and an immortal human intestinal epithelial cell line (NCM460) were donated by Guangdong Province Key Laboratory of Molecular Tumor Pathology, Southern Medical University and validated by short tandem repeat sequence analysis prior to the start of this project and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco). All cells were maintained in a cell incubator under 5% CO2 at 37 °C.
IHC was performed according to standard protocols as previously described . After deparaffinization and rehydration, 4-μm-thick tissue slides were blocked with 3% H2O2 and boiled in ethylenediaminetetraacetic acid (EDTA) (0.01 M, pH 8.0) for antigen retrieval. After blocking with 10% normal goat serum PBS, the slides were incubated with primary antibody at 4 °C, followed by treatment with secondary antibody and diaminobenzidine (DAB). Information on the primary and secondary antibodies used in this study is provided in Additional file 1: Table S1. The staining intensity of each tissue section was determined by two experienced pathologists in a double-blind manner. We referred to published standards to define “low” and “high” expression of SRSF9 . Tumor cells were graded according to the following criteria: ① Staining intensity score: 0, no staining; 1, poor staining; 2, moderate staining; and 3, strong staining; ② Positive staining score: 0, < 10% positive; 1, 10–30% positive; 2, 30–50% positive; and 3, > 50% positive. The total score was calculated as ① Staining intensity score × ② Positive staining score. Samples with total scores ≥ 5 were defined as showing high expression; those with scores < 5 were defined as showing low expression.
RNA isolation and qRT-PCR
Total RNA was extracted from human CRC cells and tissues, and mRNA was reverse-transcribed into complementary DNA using the PrimeScript™ RT reagent kit (TaKaRa, Shiga, Japan) in accordance with the manufacturer’s recommendations. qRT-PCR was performed in a 7500 Fast Real-Time PCR System with SYBR Green qRT-PCR master mix (TaKaRa). Information on the primer sequences used in this study is provided in Additional file 2: Table S2. Each experiment was performed in triplicate, and expression of all genes was normalized to that of β-actin.
Western blotting and antibodies
Protein samples were separated by electrophoresis on 10% sodium dodecyl sulfate gels and transferred to polyvinylidene fluoride membranes (Millipore, MA, USA). The membranes were then blocked in 5% skim milk for 1 h at room temperature, followed by incubation with primary antibodies for 8 h at 4 °C. The membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Finally, the signal density was detected by chemiluminescence using an ECL reagent kit (FDbio Science, Hangzhou, China). Information on the primary and secondary antibodies used in this study is provided in Additional file 1: Table S1.
Cell transfection and lentiviral infection
For transient transfection, small interfering RNAs (siRNAs) directed against SRSF9 (#SIGS0008682-4, RIBOBIO, Guangzhou, China), DSN1 (#SIGS0013609-1, RIBOBIO), METTL3 (#SIGS00056532-1, RIBOBIO) and negative control RNAs (si-ctrl) were synthesized by RIBOBIO Company (Guangzhou, China). Transient transfection was performed using a Hieff Trans™ Liposomal Transfection Reagent kit (#40802, Yeasen, Shanghai, China) in accordance with the standard protocol. Cells were collected after 24 h for qRT-PCR and after 48 h for Western blotting and functional studies.
For lentiviral transfection, Flag-SRSF9 (ov-SRSF9), empty vector (Vector), shSRSF9 (sh1, sh2), and shNC were purchased from GeneChem Company (Shanghai, China). Caco2 cells and HT29 cells were used to establish stable SRSF9 overexpression models, and HCT116 cells and LOVO cells were used in the stable SRSF9 knockdown experiments. According to the manufacturer’s instructions, 4 × 104 cells per well were seeded and transfected with the indicated lentiviruses. The infected cells were screened using 5 μg/mL puromycin (Solarbio, Beijing, China) for 1 week or longer, and transfection efficiency was determined by qRT-PCR and Western blotting analysis.
Cell proliferation assays
For CCK-8 assays, treated cells were placed in 96-well plates at 1 × 103 cells/well in quintuplicate and cultured in RPMI-1640 medium containing 10% FBS for 4 h, 8 h, 16 h, 32 h, and 64 h at 37 °C in 5% CO2. CCK-8 detection was performed using a Cell Counting Kit-8 (CCK-8, Yeasen) according to the manufacturer’s instructions. The cells were cultured in the presence of 100 μL of CCK-8 (1:10 dilution) for 2 h at 37 °C, and the optical densities (ODs) at 450 nm of the cultures were then measured. Each sample was analyzed in triplicate.
For the colony-forming assay, treated cells were seeded in 6-well plates at 200 cells/well and incubated in RPMI-1640 medium containing 10% FBS for 2 weeks at 37 °C in 5% CO2. The cells were then washed twice with PBS and fixed in 4% paraformaldehyde (Leagene, Beijing, China) for 20 min at 4 °C. Staining with 0.1% crystal violet (Sigma, St. Louis, MO, USA) was then performed within 20 min. The stained cells were counted using a scanner (Bio-Rad, Hercules, CA, USA). Each sample was analyzed in triplicate.
Transwell migration and invasion assays
Transwell chambers (Corning, NY, USA) were used to observe cell migration and invasion. For migration, cells were collected and resuspended in serum-free medium. A total of 200 µL of cell suspension at 5 × 105 cells/mL was placed in the upper chamber, and 600 µL of RPMI-1640 medium containing 10% FBS was added to the lower chamber. For invasion, the upper chamber was covered with 2 mg/mL Matrigel (Corning) prior to cell seeding. After allowing time for migration and invasion, the cells were fixed in 4% paraformaldehyde (Leagene) for 20 min at 4 °C and stained with 0.1% crystal violet (Sigma) for 30 min. The cells in three randomly selected visual fields of each sample were photographed and counted under a light microscope at 200× magnification. Each sample was analyzed in triplicate.
Wound-healing assay was also performed to observe cell migration. Monolayers of cells were uniformly scratched using the narrow edge of a 10-μL pipette tip and cultured in serum-free RPMI-1640 medium. At 24 h and 48 h thereafter, the extent of healing of the scratches was observed under a light microscope at 200× magnification. Each sample was analyzed in triplicate.
Treated cells were washed three times with PBS and fixed in 70% cold ethanol overnight at 4 °C. The cells were then washed again, centrifuged, suspended and stained with 50 μg/mL propidium iodide and 1 mg/mL RNase in PBS. Cell cycle analysis was performed using a flow cytometer (BD Biosciences, NJ, USA) and analyzed by ModFit software (BD Biosciences). Each sample was analyzed in triplicate.
In vivo tumorigenesis assay
Animal experiments were approved by the Use Committee for Animal Care and performed in accordance with institutional ethical guidelines for animal experiments. Stable Caco2 and HCT116 cells (5 × 106 cells/injection) were resuspended in 200 μL of PBS and subcutaneously injected into 4-week-old female athymic BALB/c nude mice purchased from the Guangdong Medical Laboratory Animal Center. The resulting tumors were measured every 3–5 days, and their size was calculated according to the formula: volume = 1/2 * (width2 × length). After 4 weeks, the mice were sacrificed, and the xenograft tumors were excised, photographed, and fixed in formalin for histological analysis.
Downstream target prediction
Downstream gene data for the RNA-binding protein SRSF9 were obtained from the web tool POSTAR2 (http://lulab.life.tsinghua.edu.cn/postar), which is a comprehensive database for exploring posttranscriptional regulation based on high-throughput sequencing data and provides information on a large number of binding sites for RNA-binding proteins . Data on the differentially expressed genes (DEGs) between 473 human CRC tissue sample data sets and 41 normal tissue sample data sets downloaded from The Cancer Genome Atlas (TCGA) database (https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga) were analyzed using the edge R package  of R software (Vienna, Austria). The DEGs between 17 human CRC tissue sample data sets and 17 normal tissue sample data sets obtained from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) (GSE32323) were analyzed by the limma package  of R software. Software default settings were utilized in the analyses. Downstream genes of SRSF9 identified in the POSTAR2 database and the DEGs from TCGA and GEO were screened using Venn diagrams to identify the shared genes. Potential downstream target genes had to meet the following requirements: (1) their mRNAs were binding targets of the human RNA-binding protein SRSF9; and (2) the differences in their expression between human CRC tissue samples and normal tissue samples had P values < 0.05 by statistical analysis and log2(fold change) > 1.
Survival analysis in the CRC dataset
The association of SRSF9 and DSN1 expression with overall survival was analyzed using the web tool GenomicScape (http://www.genomicscape.com/microarray/survival.php), which was established based on data obtained from GEO [21, 22]. Fifty-five CRC individuals in the GEO database (GSE17538) were sorted according to the expression of SRSF9 and DSN1. All cases from GenomicScape were provided to the algorithm for survival analysis. Kaplan–Meier survival plots with hazard ratios (HRs) and log-rank P values were obtained using the webpage. P values < 0.05 were considered to indicate significant differences.
Methylated single-stranded RNA affinity assay
Single-stranded RNA oligonucleotide probes containing the m6A-binding consensus sequence GGACU with methylated (ss-m6A, 5′-biotin-CGUCUCGG(m6A) CUCGG(m6A)CUGCU-3′) or unmethylated (ss-A, 5′-biotin-CGUCUCGGACUC GGACUGCU-3′) adenosine were synthesized by RIBOBIO Company (Guangzhou, China) and validated by mass spectrometry. According to the standardized procedure used with the RNA pull-down kit (gzscbio, Guangzhou, China), each single-stranded RNA oligonucleotide probe was immobilized on streptavidin magnetic beads and coincubated with total proteins extracted from LOVO cells for 8 h at 4 °C. After two washes, the proteins combined with the single-stranded RNA oligonucleotide probe (methylated or unmethylated) were separated by electrophoresis on 10% sodium dodecyl sulfate gels and detected by silver staining and immunoblotting analysis.
Silver staining of protein gels
Proteins that had been separated on gels were stained using a Protein Fast Silver Stain Kit (BBproExtra, Guangzhou, China) according to the manufacturer’s recommendations, and the silver signal density was analyzed using a scanner (Bio-Rad).
Gene-specific m6A qRT-PCR
Total RNA was isolated from LOVO cells. According to the standard operating protocol for the EpiQuik™ CUT&RUN m6A RNA Enrichment Kit (Epigentek, NY, USA), RNA samples were fragmented and incubated with anti-m6A antibody (#A-1801, Epigentek) for 90 min at room temperature. A nonimmune IgG was used as a negative control. RT-qPCR assays with DSN1 primers were performed to quantify the enrichment of m6A-containing RNA. Information on the primer sequences used in this study is summarized in Additional file 2: Table S2. Each experiment was performed in triplicate, and all samples were normalized to β-actin.
Dual-luciferase reporter assay
cDNAs containing the SRSF9-binding region of DSN1 mRNA sequences (chr20:36773597–36773736) were cloned into a GV361 control reporter plasmid consisting of firefly luciferase (F-luc) and verified by DNA sequencing (GeneChem, Shanghai, China). In the mutant reporter plasmid, adenosine (A) on the m6A motif was replaced by thymine (T). The GV219 vector (GeneChem) formed the backbone of the SRSF9 expression plasmid, and DNA sequencing was performed for verification. 293T cells were seeded into 24-well plates followed by cotransfection with 500 ng of DSN1 luciferase reporter plasmid (GV361-DSN1-WT and GV361-DSN1-MUT) and 0 µg, 0.25 µg, or 0.5 µg of SRSF9 expression plasmid (GV219-SRSF9) using a Dual-Luciferase Assay kit (Promega, WI, USA). After 48 h, the cells were harvested, and luciferase activity was measured according to the recommended protocols. Each group was assayed in triplicate.
Measurement of mRNA stability
Stable LOVO cells and stable Caco2 cells were incubated with 5 μg/mL actinomycin D (APExBIO, TX, USA) for 0 h, 2 h, 4 h, 6 h, or 8 h, and RNA was then extracted from the cells. Analysis of the half-life of DSN1 mRNA was performed using qRT-PCR as described earlier .
The results are presented as the mean ± SD. Student’s t test (two-tailed) and one-way or two-way ANOVA were used to evaluate quantitative data. The chi-squared test was used to analyze qualitative data. The Wilcoxon test was used to analyze rank data. The log-rank test was used to assess significant differences in overall survival. All of the analyses were performed in SPSS 24.0 (SPSS, Inc., IL, USA) and GraphPad Prism 6.0 (GraphPad, Inc., CA, USA). The level of statistical significance was defined as P < 0.05 (*P < 0.05; *P < 0.01; ***P < 0.001; ****P < 0.0001; NS: not significant).