Two variants of Interleukin-1B gene are associated with the decreased risk, clinical features, and better overall survival of colorectal cancer: a two-center case-control study

Characteristics of the study population

Baseline characteristics of the study population and their statistical significance were summarized in Table 1. The CRC patients and healthy controls were aged 56.46±11.70 and 56.00±10.59 years, respectively. The male-female ratios in the cases and controls were 1.45:1 and 1.39:1 respectively. No significant between-group difference was found in smoking, drinking or family history of cancers. Most CRC patients were rectal carcinoma or adenocarcinoma and in the TNM stage II and III. Other clinical parameters (tumor size, CEA, CA199 and differentiation) were also recorded.

Bioinformatic analysis

GEPIA database analysis based on data from The Cancer Genome Atlas (TCGA) showed the IL-1B mRNA levels in the CRC patients were significantly higher than in the controls (Figure 1). The data from the International HapMap Project and GTEx portal showed the mutant genotype of the two polymorphisms could decrease IL-1B gene expression (Figure 2). We hypothesized the IL-1B rs1143623 and rs1143634 polymorphisms conferred susceptibility to CRC by altering the IL-1B expression.

Association of IL-1B gene polymorphisms with CRC risk and clinical parameters of CRC

Table 2 shows the genotype and allele distributions of the two polymorphisms (rs1143623, rs1143634) polymorphisms in CRC cases and controls. No significant deviation from HWE was found for the either SNP in the controls. The rs1143623 polymorphism was associated with the decreased risk of CRC in the homozygous, dominant and allelic models. Similar findings were observed for the rs1143634 polymorphism, as it decreased the risk of CRC under the five models. This association remained true after the adjustment of sex and age.

Stratified analyses according to age, sex, smoking and drinking were also conducted (Table 3). The IL-1B gene rs1143623 polymorphism decreased the risk of CRC among female, but not among male. The significant findings were further confirmed among the individuals <50 years, smokers and drinkers. Subgroup analyses by age revealed the rs1143634 decreased risks in individuals ≥ 50 years but not in < 50 years. This association held true in females, smokers and drinkers.

We explored the association between IL-1B gene polymorphisms and clinical characteristics (Table 4). The CC and/or GC genotype of rs1143623 polymorphism was correlated with decreased risk for patients with tumor size ≥ 5cm, TNM stage III+IV, and rectal cancer. Moreover, the CT genotype of rs1143634 polymorphism reduced the risk of colorectal adenocarcinoma compared to other subtypes of CRC. Similar result was found in the CT+TT genotype.

Finally, a prediction model of CRC was built using GRS and used to evaluate the effects of GRS on the CRC risk in a Chinese population (Table 5). Weighted GRS indicated the risk of CRC decreased in the tertiles quartile compared with the first quartile.

Survival analysis of CRC patients with IL-1B gene rs1143634 or rs1143623 polymorphism

Considering the protective roles of the two polymorphisms in CRC patients, we aimed to explore the relationship between SNPs and the prognosis of CRC patients. The 96 CRC patients enrolled in the first year of this study were followed up for a median period of 39 months. For rs1143623 polymorphism, the Kaplan-Meier single factor analysis showed that CRC patients carrying CC genotype versus GG genotype enjoyed longer OS (HR, 0.47, 95%CI, 0.23-0.97; log-rank P = 0.041, Figure 3). However, no difference in OS was found between the CRC patients carrying IL-1B rs1143634 TT and CC genotypes (HR, 0.78, 95%CI, 0.30-2.04; log-rank P = 0.649, Figure 3).

Study subjects

A total of 527 CRC patients were recruited at the Jiangsu Provincial of Traditional Chinese Medicine and the Third Affiliated Hospital of Nantong University. The diagnosis of CRC was confirmed by histopathological examination of the biopsy. The individuals with familial polyposis, inflammatory bowel disease or metastatic carcinoma were excluded. CRC patients were staged according to the American Joint Committee on Cancer/International Union against Cancer tumor-node-metastasis (TNM) staging system. The 639 healthy controls were selected from the volunteers undergoing general health check-up in the above two hospitals. The medical and follow-up data of patients, including age, gender, lymph node metastases, TNM stage, and location and differentiation degree of primary tumor were retrieved from hospital records and interviews. As for lifestyle factors, alcohol drinker was defined as an individual consuming alcohol more than once per day for ≥3 months, while smoker was defined as smoking at least once a day for ≥1 year.

This study was approved by the institutional review broads of the two hospitals and conducted in accordance with the Helsinki declaration. Written informed consent was obtained from all participants.

Bioinformatic analysis

Data of IL-1B expression in CRC patients and healthy controls was extracted from GEPIA (http://gepia.cancer-pku.cn/index.html) [15], which is based on public data available from TCGA and is a web server for cancer and normal gene expression profiling and interactive analyses.

Genotype and mRNA expression data of the two IL-1B gene polymorphisms were cited from the International HapMap Project and GTEx portal (https://www.gtexportal.org/home/) [16], respectively.

DNA extraction and Genotyping

In brief, 2mL of peripheral blood was collected in vacuum tubes containing 5% EDTA and frozen at -80 °C until use. Genomic DNA was extracted using a DNA Purification Kit (Tiangen Biotech) according to the manufacturer’s instructions. and the concentration and purity were estimated using NanoDrop at two optical density (OD) wavelengths 260 and 280 nm. Genotyping was done by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) using a MassARRAY system (Sequenom, San Diego, CA, USA). Completed genotyping reactions were spotted onto a 384-well spectroCHIP (Sequenom) using a MassARRAY nano dispenser (Sequenom) and analyzed by MALDI-TOFMS. Genotype were called in real time on MassARRAY RT 3.1 (Sequenom) and analyzed on MassARRAY Typer 4.0 (Sequenom). For quality control, 10% of randomly-selected samples were analyzed repeatedly.

Statistical analysis

All statistical analyses were performed on the SAS package (var. 9.1.3; SAS Institute, Cary, NC, USA). Deviation from the Hardy-Weinberg equilibrium (HWE) between observed and expected frequencies among controls was investigated using goodness-of-fit Chi-square test. Differences in demographic characteristics and lifestyle-related factors between cases and controls were compared by Chi-square test (for categorical variables) and Student’s t-test (for continuous variables). The impacts of different genotypes on other parameters were further evaluated via one-way analysis of variance (ANOVA). The odds ratio (OR) and 95% confidence interval (CI) were calculated to evaluate the SNPs-associated disease risk by binary logistic analysis. The effect size of each SNP was estimated using crude ORs. The weighted genetic risk scores (GRS) was equivalent to the sum of (log OR of SNP) × (number of risk alleles carried by the individual) across two SNPs. The GRS was subsequently divided into tertiles based on the normalized value. The association between GRS and CRC risk was then calculated by binary logistic regression. Univariate survival analyses were conducted via the Kaplan-Meier method. The overall survival (OS) was defined as date from diagnosis (colonoscopy or surgery) to death or the date last known alive. P ≤ 0.05 was considered statistically significant [17].