Data collection and processing.
To investigate B3GNT6 expression and its clinical significance in colorectal cancer, three datasets including gene expression data of colorectal cancer patients were downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). Datasets GSE39582 [9], GSE103582 [10] and GSE37182 [11] involve transcriptomic mRNA expression profile of 566, 50 and 88 colorectal cancer cases with 19, 84 and 10 normal cases, respectively. Level 3 HTSeq-FPKM files, comprising 612 transcriptome profiling RNA-Seqs of 544 cases, were collected from a TCGA dataset (https://www.portal.gdc.cancer.gov/) that included information on 452 and 96 patients with colon and rectal cancer, respectively. The clinicopathological characteristics, including age, gender, clinical TNM stage, T stage, M stage and lymph node status, CIN, MMR, KRAS and braf mutation status were included. The expression level of the B3GNT6 gene in other cell lines, organs and cancers was identified in the MediSapiens IST Online database (http://ist.medisapiens.com/). The protein expression level of B3GNT6 was reviewed by using immunohistochemical-staining data provided in the Human Protein Atlas (http://www.proteinatlas.org/)
Gene set enrichment analysis (GSEA)
In order to determine the function of B3GNT6, we conducted GSEA in patients with the top 25% and bottom 25% of B3GNT6 expression in GSE39582 dataset using GSEA 4.1.0 software (https://www.gsea-msigdb.org/gsea). The annotated gene sets c2.cp.kegg.v6.0.symbols.gmt and c2.cp.biocarta.v6.0.symbols.gmt from the pathway database were selected as the reference gene set. p<0.05, |enrichment score (ES)|>0.3 and gene size≥30 were set as the cutoff criteria.
Immunohistochemistry staining of B3GNT6 protein expression in colorectal cancer patients
To investigate B3GNT6 protein expression level in colorectal cancer patients, we conducted an immunohistochemical analysis on formalin-fixed and paraffin-embedded, surgically removed colorectal cancer specimen. From February 2016 to July 2018, we collected 23 colorectal cancer tissues and paired adjacent non-tumor tissues and related clinical information from patients who underwent a radical colorectal surgery at the Department of General Surgery, Xiangya Hospital of Central South University. All tissues collected were clinically and pathologically diagnosed as colorectal cancer. Recurrent cases or adjuvant chemo- or radiotherapy recipient were excluded from our study. The differential protein expression levels of B3GNT6 in 23 colorectal cancer and paired normal tissues were measured using IHC staining as has been stated elsewhere [12]. Rabbit polyclonal anti-B3GNT6 (21291-1-AP, Thermofisher, US) was used at a working concentration of 1:100. The scores were evaluated based on staining intensity and the percentage of positive cells for each of the sections. The staining intensity was scored as follows: 0, no staining; 1, light yellow staining; 2, yellow-brown staining; and 3, deep brown staining. The percentage of positive cells was scored as follows: 0, 0~5%; 1, 6~25%; 2, 26~50%; 3, 51~75%; and 4, > 75%. The final score was calculated as follows: positive cell score × staining intensity score. The total scores were condensed into four categories: 0 for negative (−); 1–3 for weakly positive (+); 4–7 for positive (++); and 8–12 for strongly positive (+++). All patients were sorted into two groups according to the total score. High expression of B3GNT6 was defined as a detectable immunoreaction with a total score of ≥1+.
Statistical analysis.
Statistical analyses were performed using R studio (version 1.3.1056) and Graphpad Prism (Version 8.0.2). The comparison between B3GNT6 mRNA expression in colorectal cancer and normal tissues from the TCGA and GEO databases was performed using unpaired student’s t-tests. The diagnostic value of B3GNT6 mRNA expression was evaluated by the receiver operating characteristic (ROC) curve. Survival analysis was conducted using log-rank (Mantel-Cox) test. The association between clinicopathological characteristics and B3GNT6 mRNA expression levels was determined using χ2 test. All p<0.05 was considered to indicate a statistically significant difference.