2.10 Wound-healing assay
First, we harvested HCT116 cells and RKO cells (negative control and DDX10 knockdown). Then, the cells were cultured in 96-well plates. After the confluence reached 90%, we aspirated the medium of every well and used PBS to wash the wells three times. Next, we used a pipette tip to gently draw a straight line down the wells of a 96-well plate. Finally, medium was added to every well after three washes with PBS. After 0 hours, 24 hours and 72 hours, we used the Celigo platform to scan the plate and analyse the migration area.
2.11 Transwell migration and invasion assays
Transwell assays were used to assess the migration and invasion abilities of HCT116 cells and RKO cells. HCT116 cells or RKO cells (1×105) in 100 µL medium that had been cultured in serum-free medium were added to the upper chamber, and 600 µL culture medium (30% FBS) was added to the lower chamber. Then, the plate was incubated at 22°C overnight. Finally, we dyed the cells on the lower surface of the membrane to analyse cell migration. The Transwell invasion assay was carried out as described above, but Matrigel was added to the bottom of the upper chamber.
2.12 Animal studies
The animal experiment of our study used five-week-old female BALB/c nude mice (GemPharmatech, Co., Ltd., China) weighing between 16 g and 19 g. Twenty nude mice were randomly divided into two groups with 10 mice in each group. Two groups (negative control and DDX10 knockdown) of HCT116 cells (stably expressing luciferase) (2×106 cells/100 µl) were injected into the mice via the tail vein. Then, we used an in vivo imaging system (Lumina LT, Perkin Elmer, USA) to observe tumour metastasis every week. We anaesthetized the mice before performing imaging monitoring. First, the mice were intraperitoneally injected with D-luciferin (15 mg/ml) at a concentration of 10 µl/g. After 15 minutes, the mice were anaesthetized by intraperitoneal injection of 0.7% pentobarbital sodium at a concentration of 10 µl/g. After a few minutes, the mice were anaesthetized and placed under bioluminescence imaging for imaging monitoring. All mice were cultured in a specific-pathogen-free (SPF) culture environment. At the same time, we also measured and recorded the tumour size.
2.13 Mass spectrometry and protein identification
Briefly, we used a scraper to harvest RKO cells that stably expressed Flag-DDX10 and lysed the cells with lysis buffer. The reference dosage for Co-IP was 400 µl FLAG beads to collect 15,000 µg of protein. First, we used weak lysis buffer to wash the beads four times. Then, we carried out SDS-PAGE and placed the gel into Coomassie brilliant blue R250 solution for staining. Next, we collected the protein bands and used LC-MS for analysis. The original graph file (.raw) exported from Q Exactive was changed into a ".mgf " file by Proteome Discoverer 2.1 (Thermo Fisher Scientific), and then we delivered these data to the MASCOT2.6 server for database searching by built-in facilities. Then, we received the search files (.dat) that had been processed from the MASCOT server. Finally, we selected candidate peptides according to the standard of FDR < 0.01. The protein database used in this analysis was UniProt_HomoSapiens_20386_20180905.
The cBioPortal for Cancer Genomics (https://www.cbioportal.org)[32, 33] was originally developed at the Memorial Sloan Kettering Cancer Center (MSKCC). cBioPortal was established by MSKCC for molecular oncology research. In our study, we used the "colorectal adenocarcinoma (TCGA, PanCancer Atlas)" dataset to analyse the coexpression of DDX10 and RPL35. We also obtained the clinical and mutation information of DDX10 from the "colorectal adenocarcinoma (TCGA, PanCancer Atlas)" dataset.
2.15 Co-immunoprecipitation assay
First, we collected RKO cells that overexpressed DDX10 after washing them three times with PBS. Then, these cells were added to precooled cell lysis buffer. Next, the final cell lysates were sonicated and centrifuged at 140,000 rpm for 10 minutes. The protein concentration was quantified after the supernatant was resuspended. The lysates were mixed with preblocked Flag beads at 4°C overnight. After Co-IP, the Flag beads were washed as follows. First, we washed with lysis buffer three times. Then, we added 6× loading buffer and left the samples at room temperature for 10 minutes. Finally, the cell lysates were mixed with 5× sodium dodecyl sulfate (SDS) loading buffer and then boiled for 10 minutes. Proteins were eluted from the beads and separated by western blotting.
2.16 Western blot analysis
We used a 15% SDS-PAGE gel to isolate proteins, and the proteins were transferred to a PVDF membrane. The primary antibodies used in our study were mouse anti-GAPDH (1:5,000; Santa Cruz, USA), rabbit anti-RPL35 (1:300; ABCAM, UK), rabbit anti-DDX10 (1:300; Proteintech, USA), mouse anti-FLAG (1:2,000; Sigma, USA), rabbit anti-RPL18 (1:1,000; ABCAM, UK), rabbit anti-SRFBP1 (1:2,000; ABCAM, UK), rabbit anti-HNRNPU (1:2,000; Proteintech, USA), rabbit anti-nucleolin (1:1,000; ABCAM, UK), rabbit anti-PRMT5 (1:10,000; ABCAM, UK), goat anti-rabbit IgG (1:2,000; CST, USA), and goat anti-mouse IgG (1:2,000; CST, USA).
2.17 Enrichment analyses and PPI network construction
GO and KEGG analyses are important bioinformatics tools for annotating genes and researching gene functions, biological processes and signalling pathways.[34–36] In our study, we analysed the genes that were downregulated by DDX10 antibody using R version 4.0.5. The PPI network for these genes was predicted using the Search Tool for the Retrieval of Interacting Genes (STRING; https://string-db.org/) online database.
2.18 Gene set enrichment analysis (GSEA)
The RNA-seq data from the COAD and READ datasets were obtained from the TCGA portal (https://portal.gdc.cancer.gov/). Then, we divided the TCGA samples into two groups according to the expression of DDX10 and analysed these two groups of data by GSEA. Finally, we screened the results with the criterion of normalized enrichment score (NES) ≥ 1.0 and adjusted p-value < 0.25.
2.19 Tumour Immune Estimation Resource (TIMER) database analysis
TIMER (https://cistrome.shinyapps.io/timer/)[38, 39] is an interactive website offering comprehensive analysis of immune infiltration among different types of cancer. TIMER uses various immune deconvolution methods to calculate the immune infiltrate abundances. Moreover, the subscriber can obtain high-quality figures. In this work, we searched “RPL35; COAD” in the “gene module” and performed an analysis of immune infiltration.
2.20 Statistical analysis
We used Prism version 8.0 software (GraphPad Software Inc.) to analyse all the data in this study. Unpaired t tests were used to compare two groups, and one-way ANOVA was used to compare multiple groups. The screening criteria for all data was P-value < 0.05. All of the trials were repeated at least three times.