Patients and tissue specimens
The design of this study was approved by the Human Ethics Committee of China Medical University. Each subject participated in the study provided the written informed consent. The patients undergoing surgery were from the First Hospital of China Medical University between November 2012 and June 2016. We enrolled a total of 824 cases of colorectal tissue for risk study, including 276 cases of CRC and 284 adjacent non-tumor tissue (248 cases of CRC had survival time, 230 pairs had cancer tissues and its matched adjacent tissues), 202 cases of adenoma and 62 cases of anal disease; and 248 cases of CRC tissue with survival time were used for prognosis study.
We collected the tissues of CRC, which were derived from the histological results, and the collection was according to the World Health Organization standards. The TNM staging of CRC was evaluated based on the International Union Against Cancer (UICC) / United Joint Cancer Committee (AJCC) (7th edition in 2010). There were 3 cases of CRC patients that needed to be excluded: (1) patients with XP disease; (2) patients who received chemotherapy or radiation therapy before surgery; (3) patients with hereditary nonpolyposis colorectal cancer (HNPCC).
Follow-up study was conducted until April 2018. We performed prognostic analysis of 248 patients enrolled (the follow-up time was 12 to 63 months, the average survival time was 48.15 months, and there was no death). We excluded 14 patients who lacked visits in the OS analysis. Patients with the habit of smoking at least one cigarette a day for at least one year were considered to have a history of smoking. In the meantime, the study defined the drinking history as an average daily intake of at least 50 grams of alcohol for at least one year. Clinical characteristics of cancer patients included gender, age, whether smoking or drinking, tumor location, TNM stage, invasive extent, lymph infiltrative, distant metastasis, tumor deposit, perineural invasion, vessel carcinoma embolus, growth pattern, differentiation degree, maximum diameter and family history.
The tissue was fixed in formalin and embedded in paraffin, then cut into 4 μm thick sections, and the sections were mounted on glass slides. Antigen retrieval was performed after routine dewaxing. The tissue sections were washed with phosphate buffered saline (PBS, pH 7.4). Then the sections were blocked with 10% normal goat serum for 10 minutes. The expression of XPF protein was detected with mouse anti-XPF monoclonal antibody (ab-85140, 1: 200 dilution; Abcam, Cambridge, UK), and the primary antibody was used to incubate at room temperature for one hour. We spined off the primary antibody on the slice, and then used a biotinylated secondary antibody (goat anti-rabbit antibody, Fujian Maixin) to incubate the tissue for 10 minutes. The tissue was rinsed with PBS for 10 minutes. After that, we used streptavidin Biotin-biotin peroxide at a temperature of 24-27 ° C for incubating the tissue for 10 minutes, and stained with DAB (DAB-0031, Maixin City, Fujian Province, China) on a glass slide. When the tissue stain become brown (about 30 seconds), we rinsed the DAB with PBS. Finally, the slides were dehydrated, the tissue was fixed with resin and the coverslips were covered to observe the staining.
Evaluation of immunohistochemistry
Two experienced pathologists scored XPF's expression in different tissue independently, and this process followed the double-blind principle. The pathologists scored the staining intensity and staining area of XPF respectively. If there are differences in the scores of the pathologists, two pathologists will discuss and summarize the final scores. Semi-quantitative scoring criteria were used to assess the expression of XPF. Scoring standard: (1) staining intensity was classified into four levels, including 0 (no staining), 1 (light brown), 2 (brown staining), and 3 (heavy brown staining); (2) percentage of stained cells was divided into: 0(0–5); 1(6–25); 2(26–50); 3(51–75); 4(76–100%). We got the final IS (immunoreactivity score) by multiplying staining intensity and percentage of stained scores. Finally, the IS score was classified as: negative (-), score = 0; weak positivity (+), score = 1-4; medium positivity (++), score = 5-8; and strong positivity (+++), score = 9-12.
The function and regulation network of XPF by GO and KEGG analysis
STRING is a database designed to collect, score and integrate all public sources of information on protein–protein interactions. Gene ontology (GO) analysis is a major bioinformatics tool that unifies the characterization of genes and gene products through the three components of biological processes, cell composition and molecular function. Kyoto Encyclopedia of Genes and Genomes (KEGG) is a set of databases whose main purpose is to study genetic pathways, and contains information about biological pathways, genomes, chemicals and diseases. The Database for Annotation, Visualization and Integrated Discovery (DAVID; v.6.8; https://david.ncifcrf.gov/home.jsp; accessed on September 16, 2020) was applied to perform the enrichment analyses of GO and KEGG . DAVID is an online portal that provides comprehensive annotation analysis of large gene lists. GO analysis comprises groups of molecular function, cellular function and biological process. We used STRING to explore the genes closely correlated with XPF. XPF and its interacting genes were enriched and analyzed by David for GO and KEGG pathways, respectively. The ggplot2 package in the R platform (Version 3.6.3) was used to show the obtained results. Gene Set Enrichment Analysis (GSEA) is an analysis method for whole-genome expression profiling chip data, which compares genes with predefined gene sets. By analyzing the gene expression profile data, we can understand the expression status of XPF in a specific functional gene set, and whether there is some statistical significance in this expression status. We searched the expression of XPF in normal and cancerous colorectal tissues in the Oncomine database .
All the statistical analyses were conducted using SPSS 20.0 software (IL, Chicago). The difference of XPF expression between CRC and adjacent non-tumor tissues was compared by non-parametric tests. We performed nonparametric test to evaluate the relationship between XPF expression and clinicopathological parameters of CRC. Survival analysis was performed by Kaplan-Meier method. When we compared the differences between subgroups, and the log-rank test was used. To evaluate the effect of XPF expression on CRC prognosis, the Cox proportional hazard model was used, and the multivariate Cox proportional hazard model was adjusted by age, gender, TNM stage, and differentiation degree. P <0.05 was considered as statistically significant.