Association analysis of apoptosis-related gene caspase3, Integrin a subunit 1 and glutathione sulfur transferase M1 gene polymorphisms and susceptibility to gastric cardia carcinoma

Background To explore the association of polymorphisms of apoptosis-linked genes caspase3 (CASP3), integrin a subunit 1 (ITGA1), glutathione sulfur transferase M1 (GSTM1) with susceptibility to gastric cardia carcinoma (GCC). Methods From February 2016 to March 2018, selection of 113 GCC patients was as the gastric cancer (GC), and selection of 75 patients without gastric disease was as the control. Detection of CASP3, ITGA1 and GSTM1 gene polymorphisms in patients' peripheral blood was to analyze their association with GC. Division of the GC was into the good prognosis and the unpleasing prognosis in the light of the survival of patients after surgery of 3 years, and the predictable value of gene polymorphisms of CASP3, ITGA1 and GSTM1 in GCC patients was analyzed. Results CASP3 gene rs12108497 locus, ITGA1 gene rs1862610 locus and GSTM1 genotype of the GC and the control were in accord with Hardy-Weinberg equilibrium (P > 0.05); The detection rate of CASP3 gene rs12108497 locus TC/CC type, ITGA1's gene rs1862610 locus AC/AA type and GSTM1 blank type in the GC was elevated vs. the control (P < 0.05); Logistic regression analysis manifested smoking, anxiety, helicobacter pylori infection, family history of gastrointestinal tumor, combination with chronic gastric disease, CASP3 gene and GSTM1 gene polymorphism were risk factors for GC (P < 0.05); Stratification was in the light of individual smoking status, discovering that the detection rates of CASP3 gene rs12108497 locus TC/CC type, ITGA1 gene RS1862610 locus AC/AA type and GSTM1 blank type in the smoking were crucially augmented vs. the smoking (P < 0.05); The detection rates of CASP3 gene rs12108497 locus TC/CC type, ITGA1 gene rs1862610 locus AC/AA type and GSTM1 blank type in the death were augmented vs. the survival (P < 0.05); Combined detection of CASP3, ITGA1 and GSTM1 gene polymorphisms was provided with predictive value for GCC's prognosis (P < 0.05). Conclusions CASP3 and GSTM1 genes are susceptibility genes for GCC, which might be associated with the occurrence of GCC in smoking patients, and the joint detection of multiple genes is provided with predictive value for patients' prognosis.


Introduction
Gastric cardia carcinoma (GCC) is the malignant tumor that happens at the junction of esophagus and stomach, meanwhile, the patients are not presented distinct symptom in the early phase, and the symptoms covering vomiting, deglutition difficulty are manifested in the advanced stage.Presently, genetic susceptibility is also linked with the occurrence of GCC, except for external factors (1).Relevant reports have illuminated genes like GSTM1 are nearly associated with the occurrence and advancement of multiple malignant tumors (2).Glutathione-s-transferases M1 (GSTM1), a group of isoenzymes, is nearly linked with the metabolic process of exogenous compounds, which is available to catalyze the electrophilic binding of Glutathione (GSH) sulfhydryl group with carcinogenic activity generated via the metabolic activation of various enzymes, thereby forming hydrophilic substances excreted from the body, and strengthening foreign compounds' detoxification (3).Caspase3 is a proteolytic enzyme of mediating cell apoptosis, and its family members exert a critical role in the cell apoptosis (4).
Apoptosis-related gene Caspase3 (CASP3), belonging to critical member of the Caspase family, exerts a regulatory role in the initiation and execution of cell apoptosis.Integrins, a complete membrane receptor family of existing on the surface of eukaryotic cells, are available to mediate the adhesion of cells to extracellular matrix and enable cells to attach to form a whole.Integrins 1 (ITGA1), a member of the cell adhesion receptor family, is an extremely crucial receptor protein, which is the major way for cells to combine with extracellular matrix and respond, and exerts the greatly critical action in a sequence of crucial physiological and pathological processes like cell growth, malignant tumors and wound healing (5).Nevertheless, no reports have clarified association of it with GC's susceptibility so far.Consequently, this study was to explore the association of CASP3, ITGA1 and GSTM1 genes polymorphisms with susceptibility to GCC, which was linked with the occurrence and development of this disease.

Clinical Data
From February 2016 to March 2018, enrollment of 113 GCC patients was as the GC, and enrollment of 75 patients without gastric disease was as the control during the identical period.Inclusion criteria: 1.The GC met the diagnostic criteria of GCC in Standardized Diagnosis and Treatment Guidelines for Gastric Cancer (Trial) (6); 2. Age ≥ 18 years; 3. Complete cli nical data.Exclusion criteria: 4. Patients with severe cardiac, hepatic and renal abnormalities; Patients with combination of other primary malignant tumor diseases; Patients with combination of esophageal or intestinal malignant lesions; Patients with recent adoption of antibiotics or non-steroidal anti-inflammatory drugs; 5. Non-GCC patients; 6. Patients with combination of autoimmune diseases.

Clinical data collection
Inquiry of patients' general information covering age, gender, BMI, marital status, education background, family per capita monthly income, etc. was via adopting the hospital information system.BMI: 18.5-23.9kg/m 2 was normal, < 18.5 kg/m 2 or > 23.9 kg/m 2 was abnormal.Anxiety was self-rating anxiety Scale (SAS) > 50 points.light of individual smoking status, discovering that the detection rates of CASP3 gene rs12108497 locus TC/CC type, ITGA1 gene RS1862610 locus AC/AA type and GSTM1 blank type in the smoking were crucially augmented vs. the smoking (P < 0.05); The detection rates of CASP3 gene rs12108497 locus TC/CC type, ITGA1 gene rs1862610 locus AC/AA type and GSTM1 blank type in the death were augmented vs. the survival (P < 0.05); Combined detection of CASP3, ITGA1 and GSTM1 gene polymorphisms was provided with predictive value for GCC's prognosis (P < 0.05).Conclusions: CASP3 and GSTM1 genes are susceptibility genes for GCC, which might be associated with the occurrence of GCC in smoking patients, and the joint detection of multiple genes is provided with predictive value for patients' prognosis.

Prognosis grouping
Division of patients was into the death and the survival on the grounds of their survival after cure.

Observation indexes
(1) Analysis of the clinical data and CASP3, ITGA1 and GSTM1 gene polymorphisms in peripheral blood of the GC and the control was to analyze GCC's risk factors.(2) Division of the GC was into the good prognosis and the unpleasing prognosis in the light of the 3-year postoperative survival to analyze the predictive value of CASP3, ITGA1 and GSTM1 gene polymorphisms for GCC patients' prognosis.

Statistical processing
Processing of the data was via exerting SPSS22.0 software.Manifestation of the enumeration data was in %, and comparison of the difference between groups was via c 2 test.Manifestation of the measurement data was in mean ± standard deviation (SD) after normal test, and comparison of the difference between groups was via t test.Analysis of GCC's risk factors was via adopting logistic regression.Analysis of the prognostic value of CASP3, ITGA1 and GSTM1 gene polymorphisms in GCC patients was via adopting receiver operating characteristic (ROC) curve.P < 0.05 was accepted as indicative of distinct differences.

Hardy-Weinberg balance analysis of CASP3 and ITGA1 genes in the GC and the control
The genotypes of CASP3 gene rs12108497 locus and ITGA1 gene rs1862610 locus in the GC and the control were on the grounds of Hardy-Weinberg balance (P > 0.05), as presented in Table I.

Single-factor analysis of GCC's occurrence
In the GC, the proportion of patients with aberrant BMI, smoking, anxiety, helicobacter pylori (HP) infection, family history of gastrointestinal tumor, combination with chronic gastropathy, CASP3 gene rs12108497 locus TC/CC type, ITGA1 gene rs1862610 locus AC/AA type, GSTM1 gene blank type was elevated vs. the control (P < 0.05), as manifested in Table II.

Multi-factor analysis of GCC's occurrence
Logistic regression analysis elucidated smoking, anxiety, HP, family history of gastrointestinal tumor, combination with chronic gastric disease, of CASP3 and GSTM1 gene polymorphisms were risk factors for GC (P < 0.05), as manifested in Table III.

Comparison of CASP3, ITGA1 and GSTM1 gene polymorphisms in the smoking and the nonsmoking
The detection rates of CASP3 gene rs12108497 locus TC/CC type, TGA1 gene rs1862610 locus AC/AA and GSTM1 blank type in the smoking were distinctly elevated vs. the non-smoking (P < 0.05) (Table IV).

Comparison of CASP3, ITGA1 and GSTM1 gene polymorphisms of death the survival
was 66.37% (75/113).The detection rates of CASP3 gene rs12108497 locus TC/CC type, ITGA1 gene rs1862610 AC/AA type and GSTM1 blank type were augmented vs. the survival (P <0.05), as presented in Table V.

Analysis of the predicative value of joint examination of CASP3, ITGA1 and GSTM1 gene polymorphisms for GCC's prognosis
Predictive value of combined detection of CASP3, ITGA1 and GSTM1 gene polymorphisms for GCC's prognosis (P < 0.05), as manifested in Table VI and Figure 1.

Discussion
Recently, the global incidence of GC has declined promptly, but the incidence of GCC is increasing with each passing year all over the world (7).Presently, domestic epidemiological studies have illuminated the incidence of GCC has also elevated in China (8).Researches have illuminated genetic susceptibility is associated with GCC's occurrence and development (9).CASP3, a critical apoptosis-linked gene, is located in the 4q35.1 region of chromosome, and is available to be activated via CASP9 or itself cleaved into two small molecule end-peptides during apoptosis, which is available to act on mitochondriamediated internal apoptosis pathway.Relevant researches adopted Alibaba2 software to predict that this polymorphic variation might eliminate the binding site of one GATA binding protein on CASP3 gene, thereby influencing gene transcription and selective splicing.Consequently, rs12108497 locus polymorphism is nearly associated with CASP3 gene, and rs12108497 locus-mediated ASP3 gene is linked with its location in CASP3 promoter region as well (10).ITGA1, a receptor protein in the form of heterodimer, mediates the interaction between cells and between cells and cell matrix (11,12).ITGA1 is avail- able to combine with tyrosine kinase FYN via the concave protein, thereby aggregating growth factor receptor-binding protein 2 to activate Ras/ERK pathway and modulating cell proliferation.GST, a group of multifunctional proteins, is available to catalyze the binding of reduced GSH to electron-friendly harmful compounds and expel various endogenous and exogenous potentially toxic compounds from the body in a non-enzymatic form.GSTM1 is a critical enzyme of catalyzing phase reaction in GST biotransformation reaction, and the maintenance of its activity is of dramatical value for the elimination of multiple carcinogens.Several relevant researches have illuminated GSTM1 gene polymorphism is nearly associated with the occurrence of cancers like GC and uterine cancer (12,13).Based on this, this study analyzed the association of GSTM1 gene polymorphism with GCC, and the results clarified the detection rates of CASP3 gene rs12108497 locus TC/CC type, ITGA1 gene rs1862610AC/AA type and GSTM1 blank type in the GC were augmented vs. the control, manifesting that the aberrant rate of CASP3, ITGA1 and GSTM1 gene polymorphisms in GCC patients were augmented, and suggesting that CASP3, ITGA1 and GSTM1 gene polymorphisms in GC patients might be associated with GC's occurrence and advancement.Logistic regression analysis has elaborated smoking, anxiety, HP infection, family history of gastrointestinal tumor, combination with chronic gastric disease, CASP3 and GSTM1 gene polymorphisms are risk factors for GC, and the reasons are as follow: GSTM1 is available to influence the detoxification ability of individuals to environmental carcinogens, elevated ITGA1 is available to augment gastrointestinal mucosa inflammation, CASP3 is available to mediate cell apoptosis, and multi-gene interaction is available to further boost GC's occurrence (14).
Whether smoking and genetic susceptibility are provided with the combined effect in GC's advancement has also become a research hotspot among GC's risk factors.Numerous researches have clarified smoking is nearly associated with GC's occurrence (15,16).Recently, studies have illuminated smoke contains free radicals, which is available to destroy genetic genes, damage cell membranes and reduce immune function, thus leading to cancer's occurrence (17,18).Consequently, the author maintains smoking might influence GCC's occurrence via impacting gene.In this research, stratification was conducted in line with the individual smoking status, which manifested the detection rates of CASP3 gene rs12108497 locus TC/CC type, ITGA1 gene rs1862610 locus AC/AA type and GSTM1 blank type in the smoking were distinctly elevated vs. the nonsmoking, illuminating that CASP3, ITGA1 and GSTM1 gene polymorphisms in GCC patients might be associated with smoking, and the reasons are uncertain presently.Consequently, further analysis is required in the later stage.
Currently, the research has elucidated gene polymorphism exerts a certain role in GC's growth and metastasis.CASP3 is an effector of the Caspase apoptosis cascade pathway and exerts a critical action in both external and internal apoptosis (19,20).GSTM1, a member of the Mu family, is available to catalyze the initial step of GSH binding, and is the critical enzymes in biological transformation of the body, exerting a crucial role in the metabolic environment carcinogens.GSTM1 gene polymorphism is available to impact carcinogens' metabolism, and then influence cancer cells' growth (21,22), clarifying that it might be associated with patients' prognosis.The results of this study illuminated the detection rates of CASP3 gene rs12108497 locus TC/CC type, ITGA1 gene rs1862610 locus AC/AA type and GSTM1 blank type in the death were elevated vs. the survival, elaborating that CASP3, ITGA1 and GSTM1 gene polymorphisms are linked with GCC's prognosis.This research has discovered combined detection of CASP3, ITGA1 and GSTM1 gene polymorphisms is provided with predictive value for GCC's prognosis, clarifying that detection of CASP3, ITGA1 and GSTM1 gene polymorphisms is available to be adopted to early assess GCC patients' prognosis.This might be linked with the involvement of these three genes in cell advancement (23,24).
In brief, CASP3 and GSTM1 genes were susceptibility genes for GCC's occurrence, which might be associated with the occurrence of GCC in smoker patients, and the joint detection of multiple C genes was provided with predictive value for patients' prognosis.

Figure 1
Figure 1 Coordinate curves for disease activity parameters and ROC curve showing Area under the Curve (AUC) of u/NGAL, proteinuria 24h, SLEDAI/r Up/k, anti ds DNA Ab, ANA.

Table I
Hardy-Weinberg equilibrium analysis of CASP3 and ITGA1 genes in the GC and the control.Table II Univariate analysis of GCC (cases).

Table IV
Comparison of CASP3, ITGA1 and GSTM1 gene polymorphisms of the smoking with the non-smoking.

Table V
Comparison of CASP3, ITGA1 and GSTM1 genes polymorphisms in the death and the survival.Table VI Predictive value of combined detection of CASP3, ITGA1 and GSTM1 gene polymorphisms for GCC's prognosis.