2.1. Gene expression profile data
The independent COAD gene expression profiles, GSE17536, consisting of 177 COAD patients, was selected from the GEO database, whose platforms was GPL570 (Affymetrix Human Genome U133 Plus 2.0 Array). The gene transcriptome, clinical and survival information of COAD patients were obtained from TCGA database (https://portal.gdc.cancer.gov/; TCGA-COAD), which contains 470 COAD samples and 41 adjacent normal samples. Data preprocessing include background correction, data normalization, removing batch effects, combining normal and tumor group data, ID transform gene symbol, and probe supplemental missing value.
2.2. Identification of differentially expressed genes
The differentially expressed mRNA data were screened utilizing “limma” package, with threshold of log2 fold change (FC) > 1 and p-value < 0.05. Immune-related genes were obtained from the ImmPort (https://www.immport.org).
2.3. Construction of prognosis-model and nomogram.
On the basis of 436 immune-related mRNAs identified, the prognosis-related genes were primarily identified through ''survival'' package. In detail, 436 mRNA expression profile data were firstly combined with the survival information of COAD patients. Then, univariate cox analysis, LASSO regression and multivariate cox analysis were performed in sequential order to screen prognosis-related genes. The prognosis model was established with ''Risk scores = ''. The patients whose risk scores were above the median were defined as a high-risk group; otherwise, the low-risk group. ROC curve was used to evaluate the effectiveness of our prognosis model. Kaplan-Meier survival curve was used to compare the prognosis difference in high-risk group and low-risk group. P-value <0.05 was considered to be significant for the impact of OS. The nomogram was constructed based on the model established and clinical features. Calibration curves were used to measure the fitting degree of the actual OS and the predicted OS (one, three, five and eight years).
2.4. Clinical correlationanalysis
The association between clinical features (age, gender and each subset of TNM) and immune-related genes was analyzed by R software using the Wilcox test. The 7th edition of the TNM stage system 23 was adopted, and Mx was defined as unable to evaluate the presence or absence of distant metastasis.
2.5. Gene set enrichment analysis (GSEA) analysis
To further understand the underlying molecular mechanism and biological function of our model genes, GSEA was utilized with “c2.cp.kegg.v7.1.symbols.gmt” gene sets. In detail, the number of permutations was 1000; the metric for ranking genes was Signal2Noise. High risk group was regarded as experimental group and the low-risk group was seen as a reference group. FDR <0.25, and nominal P-value < 0.05 were used as the critical criterion.
2.6. Cell Lines and reagents
Four colon cancer cell clines, including SW620, LoVo, HCT116, RKO, and COLO205, and a normal colonic epithelial cell lines, HIEC-6, were utilized for the study. These cell lines were cultured in RMPI 1640 medium (Life Technologies, Gaithersburg, MD), with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) at 37°C in a 5% CO2 atmosphere.
2.7.Quantitative reverse transcription polymerase chain reaction
Total RNA was extracted from the HIEC-6, SW620, LoVo, HCT116, RKO, and COLO205 cell lines utilizing TRIzol reagent (Life Technologies). Total RNA was used as a template to synthesize complementary DNA (cDNA) utilizing PrimeScript RT Reagent Kit. Then, qRT‐PCR was conducted with SYBR Premix Ex Taq (Takara Bio Inc.). The primer sequences used for real-time PCR were listed in the Table S1. Last, the ABI 7900 system (Applied Biosystems) was used to perform qRT‐PCR assays.
2.8. Cell proliferation assay
The Cell Counting Kit‐8 (CCK‐8) assay (Vazyme Biotech Co.,Ltd) was used to assess the cell proliferation. SW620 and LoVo cells were seeded into 96-well plates (5 × 103 cells/plate). 10 microlitres (ml) of CCK-8 was added to each well of the plate at the several times: 0 h, 24 h, 48 h, 72 h and 96 h. Then, optical density (OD) was measured at 1‐4 days at a wavelength of 450 nm.
2.9. Colony formation assay
SW620 and LoVo cells were plated into 6-well plates (1 × 103 cells/plate) and cultured for 2-3 weeks. Then, cells were fixed with 10% formaldehyde for 15 minutes and stained with 1% crystal violet for 30 s prior to compute the number of colonies.
2.10. Cell invasion and migration assay
Cell invasion and migration was performed with transwell plates (24-well insert, 8 μm pore size; BD Biosciences, Bedford, MA, USA), respectively. For cell invasion assay, filters needed covered with 100 μL of Matrigel (1:5 dilution; BD Biosciences). Then, 5×104 SW620 and LoVo cells were seeded into the upper chamber with serum-free RPMI-1640. And, 600 μl RPMI-1640 medium with 10% FBS was added to the bottom chamber. After 36h of incubation , the chamber was fixed with 4% paraformaldehyde for 15 min and stained with 1% crystal violet for 30S. Last, magnification microscope was utilized to count the number of cells in the bottom chamber.
2.11. Statistical analysis
In this research, the experiments were carried out in triplicate, and the data were expressed as the mean ± standard deviation.