Abstract The aim of the present study was to investigate the matrix metalloproteinase  12 (MMP  12) gene promoter region of the transcription start point upstream 82bp at the A / G single nucleotide polymorphism (SNP) and Hebei esophageal and cardia high incidence District – Cixian the Shexian esophageal squamous cell carcinoma (ESCC) and gastric cardia adenocarcinoma (GCA), genetic predisposition relationship.  restriction fragment length polymorphism (PCR  RFLP) using the polymerase chain reaction to detect 316 ESCC patients, 243 GCA patients and 609 healthy controls MMP  12 A  82G SNP genotypes. 0.05)。">Results of MMP  12 SNP genotype and allele frequency between the two patient groups and the healthy control group, the overall distribution of differences not statistically significant (P> 0.05). Stratified analysis according to smoking status found smoking group in GCA patients with control group A / A, A / G genotype frequencies were 89.8%, 10.2% and 95.0%, 5.0%, GCA patients Group A / G genotype frequency although it is significantly higher than that in the control group, two groups showed no significant difference (χ 2 = 3.43, P = 0.06), compared with A / A genotype, portable A / G genotype have increased risk of smoking individuals GCA trend (OR = 2.13, 95% CI = 0.94 ~ 4.83). Conclusion of MMP  12 gene A  82G SNP may be nothing to do with the population of Cixian and Shexian the esophageal and gastric cardia cancer risk; carry A / G genotype may be smokers GCA incidence of risk factors.

Key words esophageal squamous cell carcinoma; cardia adenocarcinoma; MMP  12; gene polymorphism; cancer susceptibility

Correlation of MMP  12 Polymorphism to Risk of Squamous Cell Carcinoma and Gastric Cardiac Adenocarcinoma

    ZHANG Xiao  Juan, WANG Na, ZHOU Rong  Miao, DONG Xiu  Juan, LI Yan

    Division of Molecular Biology, The Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, China

    Corresponding Author: LI Yian, E  mail: lykx1962@yahoo.com.cnAbstract: Objective This study was designed to investigate the correlation of MMP  12 A  82G single nucleotide polymorphism (SNP) with susceptibility to esophageal squamous cell carcinoma (ESCC) and gastric cardiac carcinoma (GCA) in a population of high incidence region of Hebei Province. Methods MMP  12 promoter SNP (A  82G) was genotyped by polymerase chain reaction  restriction fragment length polymorphism (PCR  RFLP) analysis in 316 ESCC patients 0.05). When stratified for smoking status, the A/A">, 243 GCA patients and 609 healthy controls. Results The overall genotype and allelotype distributions of MMP  12 in ESCC and GCA patients were not significantly different from those in healthy controls (P> 0.05). When stratified for smoking status, the A / A , A / G genotype frequencies of MMP  12 in GCA patient and healthy controls of smoker group were 89.8%, 10.2% and 95.0%, 5.0% respectively, there was difference between two groups but no significant statistical difference (χ2 = 3.43, P = 0.06). And the A / G genotype frequencies of MMP  12 in GCA patients were significantly higher than those in healthy controls, compared with A / A genotype, individuals with A / G genotype have a tendency of increasing susceptibility to GCA in smoker group (OR = 2.13, 95% CI = 0.94 ~ 4.83). Conclusions In high incidence region of Hebei Province, the overall genotype and allelotype distributions of MMP  12 A  82G in ESCC and GCA patients were not significantly different from those in healthy controls; and in smoking individuals, A / G genotype probably is a risk factor for GCA.

    Key words: Esophageal squamous cellcarcinoma; Gastric cardiac adenocarcinoma; MMP  12; Polymorphism; Susceptibility

Matrix metalloproteinase (matrix metalloproteinases, MMPs) is a zinc-dependent proteolytic enzyme family, it can be an active ingredient of the degradation of extracellular matrix, regulating cell adhesion, and to maintain the tumor microenvironment play an important role in the process of tumor invasion and metastasis. On the other hand, may also regulate cell proliferation and apoptosis, change the microenvironment of the cells and modulate angiogenesis involved in tumorigenesis process [1  3]. MMP  12 is the one in this family, also known as macrophage elastase or metalloelastase, the substrate is a variety of extracellular matrix and non-extracellular matrix component. Of MMP  12 gene promoter region of the transcription start upstream 82bp at the presence of single nucleotide polymorphisms (single nucleotide polymorphism, SNP), in vitro experiments indicate here the SNP can be affected by changes in gene transcription activity of the protein expression [4 MMP  12 A  82G SNP with esophageal cancer, cardiac susceptibility relationship has not been reported.

    In this study, polymerase chain reaction  restriction fragment length polymorphism (polymerase chain reaction  restriction fragment length polymorphism, PCR  RFLP) analysis method to investigate MMP  12 A  82G SNP cardia high incidence of esophageal cancer in Hebei Province, – Cixian, and SheXian crowd of esophageal squamous cell carcinoma, gastric cardia adenocarcinoma susceptibility.

    1 Materials and Methods

    1.1 The object of study

    316 esophageal squamous cell carcinoma (esophageal squamous cell carcinoma ESCC) patients, 243 cardia adenocarcinoma (gastric cardiac adenocarcinoma, GCA) patients were from Hebei Province esophageal the cardia the high incidence Cixian and Shexian precancerous census 609 healthy controls in the same area and the same time, precancerous census, all subjects were informed consent to this study, investigating the registration of all subjects of gender, age, smoking history and family of upper gastrointestinal tumors by specialized personnel history. Currently smoking or who smoked more than five a day and last for two years or two years or more is defined as smoking individuals. The family of the one or more first-degree relatives, and (or) two or more second-degree relatives suffering from esophageal / gastric cardia / gastric defined as a positive family history of upper gastrointestinal tumors (upper gastrointestinal cancer, UGIC).

    The 1.2 specimen collection and peripheral blood leukocyte DNA extraction

    All individuals were collected peripheral venous blood 5ml, sodium citrate, 4 ℃ refrigerator. Proteinase K digestion and in the week after blood collection  salting out method [5] extracted peripheral blood leukocyte DNA.

    1.3 MMP  12 A  82G SNP genotyping type

    MMP  12 A  82G genotyping PCR  RFLP method. PCR reaction was 20μl, wherein template DNA 100ng, 10 × PCR buffer, 2μl, dyes 2μl Taq DNA polymerase (Beijing Parkson race Bio Co., Ltd.) 2.5U dNTPs 200μM upstream primer (5 ‘ GAGATAGTCAAGGG ATGATATCA  3’ each 200 nM) and downstream primer (5 ‘ AAGAGCTCCAGAAG CAGTGG  3’), MgCl2 (25 nM) 1.2 μl. PCR conditions: 94 ° C denaturation 10min, then 94 ℃ for 45s, annealing at 57 ℃ 45s, 72 ℃ extension of the 45s, 35 cycles of 72 ° C to continue extending 7min. PCR products 199bp, 4% agarose gel electrophoresis analysis of genotype by restriction endonuclease Pvu II digestion 16h (Takara Biotechnology Co., Ltd.) 37 ° C, after a 1% agarose gel electrophoresis. A / G genotype exists Pvu Ⅱ recognition sites, 199bp, 175bp and 24bp three DNA fragments by restriction enzyme digestion and performance, and the lack of A / A genotype Pvu Ⅱ, recognition sites only a 199bp fragment, as shown in Figure 1.

    For genotyping quality control, each batch of PCR reactions are sterilized double distilled water alternative template DNA was used as a negative control, and 10% DNA samples were randomly picked to repeat the experiment.

    1.4 statistical methods

    Observed value and the expected value of the chi-square test comparing genotype frequencies and Hardy  Weinberg equilibrium analysis. The chi-square test was used for the case group and the control group of of MMP  12 genotype and allele distribution was used to compare the list of lines ×. Application of non-conditional logistic regression model calculated the relative risk, odds ratio (odds ratio, OR) and its 95% confidence interval (confidence interval, CI), P <0.05 for the difference was statistically significant. Statistical analysis using SPSS11.5 software package.

    2 Results

    2.1 General characteristics

    0.05)。">ESCC patients, gender, age structure between GCA patient group and the control group was similar (P> 0.05). ESCC patients, the group of GCA patients smoking compared to the proportion of individuals with the control group showed no significant difference (χ2 = 0.53 and 2.70, P = 0.47 and 0.10). ESCC patients UGIC positive family history of the proportion of individuals in the group of GCA patients were 49.4%, 50.2%, significantly higher than that in the control group 36.0% (χ2 = 15.51 and 14.68, P value 0.00), indicating that UGIC family history may increase the ESCC and the risk of developing GCA (by gender and age corrected OR = 1.74 and 1.77, 95% CI = 1.32 ~ 2.29 and 1.31 to 2.39), are shown in Table 1.

    All specimens were successfully carried out the genotyping, all repeat typing results are consistent with the original results. 0.05)。">The chi-square test, the healthy control group, MMP  12 A  82G genotype distribution consistent with Hardy  Weinberg equilibrium (P> 0.05).

    2.2 MMP  12 A  82G SNP ESCC, GCA

    A / A, A / G and G / G genotype frequencies of MMP  12 A  82G SNP in ESCC patients, GCA patients and control groups were 96.2%, 3.8%, 0,93.4%, 6.6%, 0 and 94.1%, 5.9%, 0. MMP  12 A  82G SNP A / A and A / G genotype frequency between ESCC patients, GCA patients and control groups, the difference was not statistically significant (χ 2 = 1.89 and 0.14, P = 0.17 and 0.71) . A and G allele frequency in ESCC patients, GCA patients and control groups were 98.1%, 1.9%, 96.7%, 3.3% and 97.0%, 3.0%. A and G allele frequency between ESCC patients, GCA patients and control groups were not significantly different (χ2 = 1.84 and 0.13, P = 0.18 and 0.72), are shown in Table 1. Table 1 esophageal, gastric cardia, and the control group demographic distribution and stratified according to smoking status, smoking group in GCA patients with control group A / A, A / G genotype frequencies were 89.8%, 10.2% and 95.0 %, 5.0%, GCA patients with group A / G genotype frequency was significantly higher than that in the control group, but the difference was not statistically significant between the two groups (χ 2 = 3.43, P = 0.06). Compared with the A / A genotype, A / G genotype increased the relative risk of smoking individual risk of developing GCA OR = 2.13 (95% CI = 0.94 ~ 4.83). The smoking group ESCC patients A / A, A / G genotype frequency of 96.0%, 4.0%, and the control group, genotype frequencies were no significant differences between. Compared with the A / A genotype A / G genotype had no effect on the risk of smoking group crowd ESCC (OR = 0.78, 95% CI = 0.27 ~ 2.24). Also found no of MMP  12 gene A  82G SNP on the non-smoking group crowd ESCC, GCA risk of impact (OR = 0.55, 95% CI = of from 0.23 ~~ 1.30 and OR = 0.47, 95% CI = 0.16 to 1.39). Also in accordance with the stratified analysis of family history of cancer of the upper digestive tract, found no association between MMP  12 gene A  82G SNP ESCC, GCA incidence of risk, are shown in Table 2.

3 Discussion

    Esophageal and gastric cardia is common malignant tumors of serious harm to human health, China’s Hebei Province Cixian and Shexian high incidence area of ​​esophageal cancer, cardiac and other upper gastrointestinal tumors. Studies have shown that environmental factors are closely related to the occurrence of esophageal, gastric cardia, but people exposed to the same environmental factors are only a small number of people the disease, suggesting that genetic susceptibility differences exist between individuals, and this susceptibility differences in tumor play an important role in the development. This study shows that UGIC positive family history can increase the ESCC and GCA risk of further confirmed the genetic background may play an important role in the pathogenesis of this tumor, but also prompted the crowd with upper gastrointestinal cancer family history, should be regularly checks to detect early lesions.

    MMPs is highly dependent on a family of proteolytic enzymes in zinc and tumor development, invasion and metastasis. Members of most MMPs expression and activity increased with the stage of progress in almost all human malignancies, invasive enhanced and shortened survival [1,6]. MMP  12 capable of degrading a variety of extracellular non extracellular matrix protein components [7], on the one hand, it is generated by decomposition of plasminogen and Ⅴ, Ⅹ Ⅲ collagen angiostatin and endostatin inhibits angiogenesis [1, The 8  9], on the other hand, it can decompose the structural components of the extracellular matrix, such as collagen type IV and fibrin promote angiogenesis. Has been reported in some tumors, increased expression of MMP  12 the patient has a good prognosis [1].

    MMP  12 A  82G polymorphism located in the promoter region of MMP  12 gene transcription factor actin  1 (AP  1) binding region vitro experiments showed that the A allele AP  1 high binding capacity lead to MMP  12 promoter activity increased so that the increased gene expression [4]. The statistical results of this study show of MMP  12 gene A  82G SNP Hebei Province, magnetic, the Shexian crowd of esophageal and cardia cancer risk; compared with A / A genotype, portable A / G genotype increased risk of smoking individuals GCA trend (OR = 2.13, 95% CI = 0.94 ~ 4.83). Kader, etc. [10] reported that G allele significantly increase the transfer capacity of the the smoking crowd bladder cancer (OR = 2.32, 95% CI = 1.30 ~~ 4.12), Su et al [11] also study the G allele trend (OR = 1.20, 95% CI = 0.91 ~~ 1.59) increase in the male population, the incidence of lung cancer risk. Because of the smokers in the study were male, the findings of the above studies have consistency. The results of this study in genotype frequency distribution of G / G genotype is 0, and Zhang Rong Bao et al [12] study of the Chinese Han population and Shin et al [13] on the of MMP  12 in a study of women with breast cancer in Shanghai, China A  82G SNP genotyping results. G allele frequency in the study population was very low (3.0%), of this genotype uneven distribution will reduce the statistical power, part of our results is the basis of small sample size in the stratified analysis expand the sample size, and therefore need to be confirmed by further studies of this trend on the resulting. Yang et al [14] reported that MMP  12 overexpression in colorectal prolong patient survival and reduce tumor blood vessels to form, Similarly, Kerkel , etc. [15] The study also showed that MMP  12 giant tumor the expression in macrophages is associated with a well-differentiated tumor cells. These studies show and MMP  12 possible through the decomposition of plasminogen and Ⅴ type XIII collagen type angiostatin and endostatin inhibits angiogenesis play a protective role in carcinogenesis [16-17]. Therefore, we hypothesized that the promoter region upstream 82bp at the genotype is A / G MMP  12 gene than genotype A / A, reducing the possible protective role of MMP  12 weakened, thereby increasing the individual risk of developing GCA.

    The results of this study show that MMP  12 A  82G SNP is only related with the smoking crowd GCA incidence trends, the results confirmed that further studies are needed. Synergy which the presence of this polymorphism and smoking or other environmental and genetic factors, and its biological mechanism has yet to be to continue to study with ESCC, GCA patients with the degree of tumor infiltration and metastasis recurrence and prognosis.