Functional BCL-2 regulatory genetic variants contribute to susceptibility of esophageal squamous cell carcinoma

B-cell lymphoma-2 (BCL-2) prevents apoptosis and its overexpression could promote cancer cell survival. Multiple functional BCL-2 genetic polymorphisms, such as rs2279115, rs1801018 and rs1564483, have been identified previously and might be involved in cancer development through deregulating BCL-2 expression. Therefore, we examined associations between these three polymorphisms and esophageal squamous cell carcinoma (ESCC) susceptibility as well as its biological function in vivo. Genotypes were determined in two independent case-control sets consisted of 1588 ESCC patients and 1600 controls from two regions of China. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression. The impact of the rs2279115 polymorphism on BCL-2 expression was detected using esophagus tissues. Our results demonstrated that the BCL-2 rs2279115 AA genotype was significantly associated with decreased ESCC risk compared with the CC genotype (OR = 0.72, 95% CI = 0.57–0.90, P = 0.005), especially in nonsmokers (OR = 0.42, 95% CI = 0.29–0.59, P = 0.001) or nondrinkers (OR = 0.44, 95% CI = 0.32–0.62, P = 0.002). Genotype-phenotype correlation studies demonstrated that subjects with the rs2279115 CA and AA genotypes had a statistically significant decrease of BCL-2 mRNA expression compared to the CC genotype in both normal and cancerous esophagus tissues. Our results indicate that the BCL-2 rs2279115 polymorphism contributes to ESCC susceptibility in Chinese populations.

Scientific RepoRts | 5:11833 | DOi: 10.1038/srep11833 stimuli and its overexpression could promote cancer cell survival 5 . The discovery of BCL-2 established a new paradigm in cancer biology, namely that apoptosis defects give cells selective survival superiority 5 .
As one of the most common and fatal malignancies worldwide, esophageal squamous cell carcinoma (ESCC) shows a relatively high incidence in Asian including China 6 . Cigarette smoking, heavy ethanol consumption, micronutrient deficiency as well as dietary carcinogen exposure have been identified as main environmental etiological factors of ESCC 7,8 . Accumulated evidences indicate that genetic makeup may also contribute to ESCC susceptibility. For example, ESCC genome-wide association studies (GWAS) highlight the involvement of single nucleotide polymorphisms (SNP) in cancer development, alone and in combination with environmental risk factors [9][10][11][12][13][14] .
In ESCC, BCL-2 plays its role in regulating cancer cell growth, especially in the early stage. Additionally, BCL-2 expression has been positively associated with cancer cell differentiation and inversely with disease progression 15 . There are multiple functional genetic polymorphisms have been identified in the BCL-2 gene locus which is located on chromosome 18q21.3 and consists of three exons and two promoters. These two promoters show different functional properties. That is, BCL-2 mRNA transcription is driven by the P1 promoter, while the P2 promoter acts as a negative regulatory element 16,17 . There is a functional rs2279115 (− 938 C > A) promoter SNP in the inhibitory P2 promoter 18 . Interestingly, the rs2279115 A allele may render a better interaction with TP53, leading to a decrease in the BCL2 expression, an up-regulated programmed cell death or reduced longevity of transformed cells, and thus a subsequent decrease in the risk of malignances, such as squamous cell carcinoma of the head and neck (SCCHN) 19 . There is only one study with a relatively small sample size investigated the role of this SNP in the etiology of ESCC without genotype-phenotype association investigations 20 . Considering the importance of BCL-2 in tumorigenesis, we hypothesized that the BCL-2 functional polymorphisms (rs2279115, rs1801018 and rs1564483) might be also involved in ESCC development through deregulating BCL-2 expression. To test this hypothesis, we conducted a two-stage case-control study of ESCC. To validate the biological function of BCL-2 rs2279115 genetic variant in vivo, we detected the association between its genotypes and BCL-2 mRNA expression levels in normal and cancerous esophagus tissues.

Materials and Methods
Study subjects. Two case-control sets were included in the current study. Huaian case-control set consists of 588 ESCC cases from Huaian No. 2 Hospital (Huaian, Jiangsu Province, China) and sexand age-matched 600 healthy controls. Jinan case-control set contains 540 patients with ESCC from Shandong Cancer Hospital, Shandong Academy of Medical Sciences (Jinan, Shandong Province, China) and sex-and age-matched (± 5 years) 550 controls. We used the group match considering sex-and age-match between cases and controls. The detailed information about the two case-control sets has been reported previously [21][22][23] . Twenty-nine ESCC tissues and twenty nine paired esophagus normal tissues adjacent to the tumors were obtained from surgically removed specimens of patients in Huaian No. 2 Hospital. The normal tissues sampled at least 2 cm away from the margin of the tumor. All subjects were ethnic Han Chinese. This study was approved by the Institutional Review Boards of Huaian No. 2 Hospital and Shandong Cancer Hospital, Shandong Academy of Medical Sciences. At recruitment, the written informed consent was obtained from each subject. The methods were carried out in accordance with the approved guidelines.
Genotyping of BCL-2 polymorphisms. Three BCL-2 candidate SNPs were analyzed by the MassArray system (Sequenom Inc., San Diego, California, USA). A 5% blind, random sample of study subjects was genotyped in duplicates and the reproducibility was 98.8%. To reduce the costs of the study, we genotyped the BCL-2 rs2279115 SNP in the validation set using PCR-based restriction fragment length polymorphism (RFLP). The genotyping primers used for amplifying DNA segments with the SNP site were 5′ -GCATTTGCTGTTCGGAGTTT-3′ and 5′ -TTCGCAGAAGTCCTGTGATG-3′ . The 25 μ L PCR reaction mixture contains 0.2 mmol/L of deoxynucleoside triphosphate, 0.1 mmol/L of each primer, 100 ng of DNA, 1.0 U of rTaq DNA polymerase (TaKaRa), 1.5 mmol/L MgCl 2 , and 1 × reaction buffer. The PCR profile included an initial 2 minutes melting step at 95 °C, followed by 35 cycles of 30 seconds at 94 °C, 30 seconds at 60 °C, 30 seconds at 72 °C, and a final 10 minutes elongation step at 72 °C. Restriction enzyme BccI (New England Biolabs) was used to distinguish the rs2279115 C > A genotypes. A 15% random sample was reciprocally tested by different person, and the reproducibility was 99.0%.
Real-time Analysis of BCL-2 mRNA. Total RNA was extracted from ESCC tissue samples using TRIzol Reagent (Invitrogen) and converted to cDNA using the ReverTra Ace qPCR RT Kit (TOYOBO). BCL-2 mRNA expression in cancerous and normal esophagus tissues was examined using the SYBR-Green real-time quantity PCR (qPCR) method as described previously [23][24][25] . Gene expression for BCL-2 and β-actin as an internal reference gene was carried out using the ABI 7500 real-time PCR system in triplicates. The primers used for BCL-2 were 5′ -TCGCCCTGTGGATGACTGA-3′ and 5′ -CAGAGACAGCCAGGAGAAATCA-3′ ; and for β-actin were 5′ -GGCGGCACCACCATGTACCCT-3′ and 5′ -AGGGGCCGGACTCGTCATACT-3′ . The samples size of each qPCR assay is 10 μ L. Relative gene quantitation for BCL-2 was calculated by -delta delta ct methods. To control quality of the qPCR data, we repeated the qPCR assays for some specific sample if the ct values of the triplicates for this sample Scientific RepoRts | 5:11833 | DOi: 10.1038/srep11833 showed variations more than 0.5 in very few cases. Paired t-test was used to calculate the differences between individuals with different genotypes.

Statistics. The differences in demographic variables and genotype distributions of BCL-2 SNPs
between ESCC cases and controls were examined using Pearson's χ 2 test. Unconditional logistic regression model was utilized to estimate associations between BCL-2 genotypes and ESCC risk by odds ratio (OR) and their 95% confidence intervals (CIs). During calculating associations between functional SNP candidates in BCL-2 and ESCC risk in Huaian case-control set, we used the common genotypes of rs2279115 (CC), rs1801018 (AA) and rs1564483 (GG) as the reference genotype. All ORs were adjusted for age, sex, drinking and smoking status, where it was appropriate. A P value of less than 0.05 was used as the criterion of statistical significance, and all statistical tests were two-sided. All analyses were performed with SPSS software package (Version 16.0, SPSS Inc., Chicago, IL).

Results
There were no statistically significant differences between cases and controls for both case-control sets in terms of median age and sex distribution (all P > 0.05), which indicated that the frequency matching of age and sex was adequate (Table 1). More smokers were observed among ESCC cases compared with controls in both case-control sets (Huaian set: 74.3% vs. 33.8%, P < 0.001; Jinan set: 75.2% vs. 39.6%, P < 0.001). Similarly, there were more alcohol drinkers among patients than among control subjects in these two sets (Huaian set: 56.8% vs. 40.3%, P < 0.001; Jinan set 55.3% vs. 40.1%, P < 0.001).
The genotype frequencies of BCL-2 candidate SNPs (rs2279115 C > A, rs1801018 A > G and rs1564483 G > A) are summarized in Table 2. The allele frequencies for rs2279115 C, rs1801018 G and rs1564483 A were 0.362, 0.084, and 0.375 in ESCC cases and 0.410, 0.098, and 0.338 in control subjects in Huaian training case-control set. All observed genotype frequencies in either controls or cases conform to Hardy-Weinberg equilibrium. Distributions of the rs2279115, rs1801018 and rs1564483 genotypes were then compared among patients and controls. Frequencies of rs2279115 CC, CA, and AA genotypes among ESCC cases differed significantly from those among controls (χ 2 = 8.68, P = 0.013, df = 2), with the frequency of AA homozygote being significantly lower among patients than among controls (13.7% vs. 15.0%). However, no statistically significant differences of rs1801018 and rs1564483 genotypes were observed between cases and control subjects (both P > 0.05) ( Table 2). Therefore, we did no other analyses of these two polymorphisms in the next studies.
Because tobacco smoking and alcohol drinking are both risk factors for ESCC, we then examined whether the BCL-2 rs2279115 genetic variant influence ESCC susceptibility in combination with these pathogenic factors (Table 4). In nonsmokers, compared with the rs2279115 CC carriers, individuals with CA or AA genotype had a 0.70-fold or 0.42-fold decreased risk to develop ESCC (95% CI = 0.54-0.92, P = 0.010, or 95% CI = 0.29-0.59, P = 0.001). There was no significantly decreased risk for smokers with CA or AA genotype compared with CC smokers (both P > 0.05). Nondrinkers carrying rs2279115 CA  or AA genotype showed significantly decreased risk to develop ESCC compared with CC carriers who did not drink (OR = 0.78, 95% CI = 0.61-0.99, P = 0.041, or OR = 0.44, 95% CI = 0.32-0.62, P = 0.002). However, there were no association between rs2279115 CA and AA genotypes and ESCC risk in drinkers (both P > 0.05) ( Table 4).
Due to rs2279115 C-to-A change could influence BCL-2 P2 promoter activity and gene expression in cancer cells, we investigated whether there is an allele-specific effect of rs2279115 SNP on BCL-2 expression in esophagus tissues. We found that there were significantly lower BCL-2 mRNA levels (mean ± SE) among carriers of the rs2279115 CA and AA genotypes compared to carriers of the CC genotype in normal esophagus tissues (0.083 ± 0.012 [n = 17] vs. 0.109 ± 0.021 [n = 12], P = 0.031) ( Table 5). Similar results have also been observed in ESCC tissues (the rs2279115 CA and AA genotypes: 0.153 ± 0.022 [n = 17] vs. the CC genotype: 0.184 ± 0.045 [n = 12], P = 0.040) ( Table 5).

Discussion
In the current study, we investigated the association between three BCL-2 functional candidate SNPs and ESCC susceptibility through a case-control approach. We found that only BCL-2 rs2279115 polymorphism is significantly associated with decreased ESCC susceptibility in Chinese populations, with the rs2279115 AA genotype as the protective genotype. Genotype-phenotype correlation studies demonstrated that subjects with the rs2279115 CA and AA genotypes had a statistically significant decrease of BCL-2 mRNA expression compared to the CC genotype in both normal and cancerous esophagus tissues. Our data support the hypothesis that SNPs in gene expression regulatory elements of tumor suppressor genes or oncogenes might impact genetic susceptibility of cancers.
The BCL-2 rs2279115 polymorphism has been extensively studied in multiple cancer types, including ESCC. Liu et al. reported in a case-control study conducted in western China including 205 esophageal cancer patients and 224 controls 20 . They found that the BCL-2 rs2279115 AA genotype was significantly associated with increased risk of developing esophageal cancer. In contrast, we found that subjects with   Considering the distinct etiology and clinical behaviors of these two subtypes of esophageal cancer, we believe that mixed study subjects might also lead to the discrepancy. Our results are consistent to functional relevance of rs2279115 polymorphism in SCCHN 19 . That is, the rs2279115 AA genotype may result in decreased BCL-2 expression, elevated apoptosis rates of cancer cells, and thus decreased risk of malignances 19 . Song et al. recently reported that hierarchical clustering analyses of whole-genome sequencing in 17 ESCC cases and whole-exome sequencing in 71 cases indicate that ESCC and HNSCC mutation spectra were intermingled, whereas esophageal adenocarcinomas were clearly distinctive from ESCC 26 . Therefore, it is biologically plausible that the functional BCL-2 rs2279115 polymorphism influence ESCC genetics thoroughly through regulating BCL-2 expression and apoptosis in vivo.
The BCL-2 rs2279115 polymorphism showed a consistent association with ESCC risk in two independent case-control cohorts. Additionally, our results are unlikely to be attributable to unknown confounding factors due to having relatively large sample sizes, significantly increased odd ratios with small P values. More importantly, our results on the genotype-phenotype relationship between the rs2279115 polymorphism and gene expression supports our conclusion. However, there might be several limitations in the current case-control study. For instance, since all ESCC cases were recruited from the hospital, inherent selection bias may exist. Therefore, the findings of our study warrant to be validated in a population-based prospective study in the future. In addition, relatively small sample size for non-smokers and non-drinkers should be further analyzed in a larger population.
In conclusion, we demonstrated that functional BCL-2 rs2279115 SNP was associated with a significantly decreased risk of ESCC in Chinese populations, especially in nonsmokers or nondrinkers. Our data may support the hypothesis that genetic variants can influence gene regulation might be important modifiers of ESCC susceptibility. These results may lead to better understanding of ESCC etiology in different populations.