Abstract
Xianyou county of Fujian province, located on the southeast coastal of China, has higher gastric cancer mortality. Chronic inflammation plays an important role in the occurrence of gastric cancer, in which the nuclear factor-κB signaling pathway of the inflammatory reaction begins and plays an important role in the amplification process. Studies have found that a single-nucleotide polymorphism of nuclear factor-κB signaling pathway molecules encoding genes is associated with gastric cancer, but the combined effect of the nuclear factor-κB signaling pathway gene has not been explained nor has been cardia and non-cardia gastric cancer risk factors and genetic susceptibility loci. New gastric cancer cases of the Fujian Xianyou Hospital were the research object. They were divided into cardia and non-cardia cancer in order to study a single-nucleotide polymorphism of the nuclear factor-κB signaling pathway important node molecules P50 and I kappa B encoding genes NFKB1 and NFKBIA by desorption ionization time of flight mass spectrometry analysis and by matrix-assisted laser mass spectrometry. The results showed that NFKB1 and NFKBIA single-nucleotide polymorphisms and gastric cancer are related and that the combined effects of polymorphisms in two genes and the NFKBIA gene monomer increased the risk of gastric cancer, and it was found that in different types of gastric cancer (the cardia and non-cardia cancer), susceptible polymorphism sites and combined effects are different.
Keywords
Introduction
Gastric cancer is one of the most common malignant tumors of the digestive tract. Fujian Province is a high-incidence area of gastric cancer in our country. A 2011–2012 survey of Xianyou Country by our group found that gastric cancer mortality due to malignant tumors was the highest, 54.14/10 million, 1 higher than the national average (21.9/10 million). 2 The pathogenesis of gastric cancer is not clear. In recent years, most studies suggested that chronic inflammation plays an important role in gastric carcinogenesis and progress. The nuclear factor-κB (NF-κB) signaling pathway is a major regulator of inflammation. It plays a crucial role in the beginning and progress of inflammation. 3 In a study by Fu et al. 4 , it was found that NF-κBs are activated in most cancers, the equivalent of “non classical oncogene.” 4 NF-κB includes two protein family, the NFκB family and IκB family. The signal system through the two NF-κB dimers and IκB combined and separately mediate signal transduction to cause a series of gene expressions, including an immune response mediated by the cell adhesion factor, inflammation and apoptosis, cell-cycle regulation, cell differentiation, and other physiological and pathological processes, involved in the occurrence and development of gastric cancer.5,6
The occurrence of gastric cancer is the result of the interaction of environmental and genetic factors. However, the result of exposure to common environmental factors in people suffering from gastric cancer is not the same, and an individual’s genetic background to a great extent determines the risk of cancer. The single-nucleotide polymorphism (SNP) is one of the most common heritable variations in the human genome, accounting for more than 90% of all known polymorphisms. Genetic studies found that polymorphisms of genes encoding inflammatory mediators are related to the increase in gastric cancer risk, 7 indicating that genetic variation in the carcinogenesis plays an important role in gastric cancer occurrence.
The two most common dimers are P50 and p65 in NF-κB signaling pathway. The two dimers function through combination and separation with IκB. The NFKB1 gene encoding P50, NFKBIA gene encoding IκB, the rs696 and rs2233406 is 3′-UTR region and promoter region polymorphism respectively. NFKBIA gene functional analysis (http://snpinfo.niehs.nih.gov/) showed that these two sites are potential functional sites and that rs3755867 is the mutation of the NFKB1 gene intron. It was found that the mutation of this site is associated 8 with female breast cancer, but the correlation with gastric cancer has not been reported. It was also found that different areas (cardia and non-cardia gastric cancer) not only have different environmental risk factors,9,10 but also the genetic variability of the same gene is different. 11 This research aims to study the correlation between the three sites and the gastric cancer risk in the Chinese population and to explore the three loci for similarities and differences in the genetic background between cardia and non-cardia gastric cancer.
Objects and methods
Research objects
Case selection: a case-control study was used. The cases were from the hospital of Xianyou County, Fujian Province, China. The sample size of case group was 313.
The inclusion criteria were as follows: tissue samples were obtained from surgical or endoscopic examination, and the newly diagnosed cases of gastric cancer were pathologically diagnosed. Patients with benign gastric lesions and other malignant tumors were excluded.
Population control selection: according to gender, cases with the age of ±3 years were paired 1:1, with those with no cancer history and no cancer cases of immediate family members as a healthy control. The sample size of control group was 313.
Genotyping
Before treatment, 5 mL blood samples were collected and blood samples were extracted by the red blood cell lysis method. DNA was detected by the concentration of NanoDrop™ ND-1000, and the 260/280 ratio was used to determine the purity of the DNA.
DNA samples were tested by the Capital Biotechnology Company. The primer design is shown in Table 1. The detection process was Sequenom MassARRAY SNP combined with multiple polymerase chain reaction (PCR) technology, MassARRAY iPLEX single-nucleotide extension technique, and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF).
Primer sequence of each SNP locus.
SNP: single-nucleotide polymorphism; PCR: polymerase chain reaction.
Laboratory quality control
Interpretation of the genotype used the blind method. The reader did not know whether the samples were from the case group or control group. In addition, 2% of samples were randomly selected for duplicate detection by the blind method. A few samples due to DNA quality or concentration failed to detect the genotype and were removed.
Statistical analysis
For data analysis, IBM SPSS20.0 software was used. The χ2 test was used in the control group to calculate whether genotype distribution was in accordance with the Hardy–Weinberg equilibrium. It was also used for the general demographic characteristics of the equilibrium test and for distribution frequency of the three loci in each genotype and allele in the case group and the control group. Cox regression analysis was used to calculate the odds ratio (OR) value of the polymorphic loci genotype in paired case-control study, analysis the risk of gastric cancer in different genotypes, the OR value, and 95% confidence interval (CI). The SHEsis (http://analysis.bio-x.cn/myAnalysis.php) haplotype was constructed by online software and provided the evaluation data and the haplotype and gastric cancer OR value and 95% CI. All statistical tests were two-sided probability tests, and the significance level a = 0.05.
Results
The demographic characteristics and the balance test of the case group and the control group
This investigation included gastric cancer patients and population controls. There were 313 patients with gastric cancer, including 240 males and 73 females, and a similar number for the population controls. The demographic data were of the case group and the control group of contingency table χ2 test, and the results showed that except for education (χ2 = 14.04, p = 0.01), other factors were comparable (p > 0.05).
Hardy–Weinberg genetic equilibrium test
In testing the three polymorphic loci of the Hardy–Weinberg genetic balance, we found that the distribution of the three polymorphic loci in 313 population controls accorded with the Hardy–Weinberg genetic equilibrium; the χ2 value and p value were 2.047 and 0.152 for rs696 site, 0.601 and 0.438 for rs2233406 site, and 0.517 and 0.472 for rs3755867 site, and it showed that the object was a better representation of the population.
The relationship between gene polymorphisms and gastric cancer susceptibility
Analysis of gene polymorphism and susceptibility to gastric cancer
As shown in Table 2, all cases were divided into cardia cancer and non-cardia cancer. We used conditional logistic regression analysis on the three loci. The results showed that rs3755867 (GG) was associated with gastric cancer and it increased the risk of gastric cancer (OR = 1.58). rs696 (AA) was associated with cardia cancer and it increased the risk of cardia cancer (OR = 2.23). rs2233406 (CT) and rs2233406 dominant modes (CT + TT) were associated with non-cardia cancer and they increased the risk of non-cardia cancer (OR values were 1.66 and 1.65, respectively).
Analysis of gene polymorphism and susceptibility to gastric cancer.
aOR: adjusted odds ratio; CI: confidence interval; SNP: single-nucleotide polymorphism.
p < 0.05.
Analysis of the gene polymorphism combined effect
We took three gene polymorphisms of rs696, rs2233406, and rs3755867 in gene NFKBIA and gene NFKB1 to analyze the combined effect. We constructed new variables—rs696_rs2233406, rs696_rs3755867, and rs2233406_rs3755867—and assigned new variables, 0 stood for not carrying the adverse genotype, 1 stood for carrying 1 adverse genotype, and 2 stood for carrying 2 adverse genotypes.
As shown in Table 3, the combined effect analysis showed that when carrying the NFKBIA gene polymorphism site of rs696 (AA) and NFKB1 gene polymorphism site of rs3755867 (GG), the risk of cardia cancer occurrence was higher than when not carrying the adverse genotype (OR = 5.22), and when carrying the NFKBIA gene polymorphism site of rs2233406 (CT) or NFKB1 gene polymorphism site of rs3755867 (GG), the risk of non-cardia cancer occurrence was higher than when not carrying the adverse genotype (OR = 1.62).
Analysis of the gene polymorphism combined effect and susceptibility to gastric cancer.
aOR: adjusted odds ratio; CI: confidence interval; SNP: single-nucleotide polymorphism.
p < 0.05.
Haplotype analysis of the NFKBIA gene
We used SHEsis software to analyze the relationship between the NFKBIA gene haplotype and gastric cancer. NFKBIA gene polymorphism sites of rs696 and rs2233406 had high linkage disequilibrium (D′ = 0.741, r2 = 0.07). As shown in Table 4, the risk of gastric cancer occurrence increased 2.46 times in haplotype A_T (95% CI: 1.19, 5.12), and the risk of non-cardia cancer occurrence increased 6.05 times (95% CI: 1.74, 21.10).
Frequency and risk of haplotype distribution of the NFKBIA gene in gastric cancer patients and controls.
aOR: adjusted odds ratio; CI: confidence interval.
p < 0.05.
Discussion
This research used Sequenom MassARRAY SNP detection technology to detect gastric cancer patients and population controls, the genotype in the gene NFKBIA rs696 locus, rs2233406 locus, and gene NFKB1 rs3755867 locus. We found that the gene NFKB1 rs3755867 locus was associated with gastric cancer susceptibility in the Chinese population (OR = 1.58), and the combined effects analysis found that carrying the NFKB1 rs3755867 site or NFKBIA rs2233406 site, adverse genotype would increase the risk of gastric cancer occurrence (OR = 1.38). Haplotype analysis showed that the risk of gastric cancer occurrence was higher in NFKBIA rs696 site and rs2233406 site A_T haplotype (OR = 2.46).
In order to make a further stratified analysis, we divided the cases according to the pathological type of cardia cancer and non-cardia cancer. We found that the NFKBIA rs696 site was associated with the susceptibility of cardia cancer in the Chinese population (OR = 2.23), and the combined effect analysis showed that when carrying the NFKBIA rs696 site and NFKB1 rs3755867 site of the adverse genotype, the risk of gastric cancer occurrence was much higher than when not carrying the adverse genotype (OR = 5.22), but we did not find this correlation in the analysis of non-cardia cancer. It indicated that mutations of these two loci would significantly increase the risk of cardia cancer occurrence.
We also found that the NFKBIA rs2233406 mutation was associated with the susceptibility of non-cardia cancer in the Chinese population, with heterozygous mutation increasing the risk of non-cardia cancer by 1.66 times,and that the dominant genetic model (CT + TT) increased the risk of non-cardia cancer occurrence (OR = 1.65). The combined effects analysis demonstrated that carrying the NFKBIA rs2233406 site or NFKB1 rs3755867 site, adverse genotypes would increase the risk of gastric cancer occurrence (OR = 1.62).
NFKBIA is a gene encoding the IκB protein, and the IκB protein can inhibit the NF-κB protein complex. The NFKBIA gene rs2233406 polymorphism has been proved to be associated with many diseases. A recent study of the Malaysian population found that the rs2233406 polymorphism was associated with the susceptibility of sporadic colorectal cancer and that the heterozygous genotype (CT genotype) could reduce the risk of colorectal cancer in women but not in men. 12 Subsequently, the research found that carrying the heterozygous genotype (CT genotype) could reduce the risk of breast cancer in premenopausal women (OR = 0.63). 13 Research on NFKBIA 3′-UTR of rs696 polymorphism has not been limited to chronic diseases. Recently a study on reproductive health in china found that carrying the AA genotype increased the risk of defective spermatogenesis in the non smoking population. 14 The present study found that the rs696 polymorphism AA genotype was an adverse genotype of cardia cancer, and that the rs2233406 polymorphism CT genotype was an adverse genotype of non-cardia cancer. The study results were different from previous studies, showing that different tumors have different susceptibility genotypes. However, we found by stratified analysis that cardia cancer and non-cardia cancer had different susceptibility loci.
P50 is a family member in NF-kB multiple transcription factor gene NFKB1 encoding the P50 protein which is located on human chromosome 4q24. Some studies found that the NFKB1 gene polymorphism was related to gastric cancer and influenced invasion and lymph node metastasis of gastric cancer.15,16 This study found that the NFKB1 intron area of rs3755867 polymorphisms of the GG genotype was associated with the occurrence of gastric cancer in the Chinese population, but the research on this site is limited, while the site mutation was found to be related to the occurrence of breast cancer in a previous study, 8 and played a role in the CHIEF pathway. However, the mechanism has not been reported, and maybe the mutation caused splicing signal changes in eukaryotic genes, leading to the intron presenting as a hidden exon in eukaryotic RNA, encoding without an active protein, and resulting in abnormal expression of the downstream gene which is regulated by NF-kB.
In this study, we found that NF-kB signaling pathway key node SNPs were related with gastric cancer and that cardia cancer and non-cardia cancer had different susceptibility loci. Further haplotype analysis found that the risk of gastric cancer was different in different NFKBIA gene haplotypes and that carrying the mutant haplotype had a higher risk. This study began with the NF-kB signal pathway SNP mutation, found susceptibility loci and haplotypes in cardia cancer and non-cardia cancer, and provided scientific data for the study of the molecular mechanism of gastric cancer.
Footnotes
Acknowledgements
The authors thank Dr Edward C. Mignot, Shandong University, for linguistic advice.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Natural Science Foundation of Fujian Province, China (Grant No. 2015J01673; 2017J01811), Project of etiology and epidemiology research on digestive tract tumor in Xianyou County of Fujian Province, China (Grant No. 2013B008).
References
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