Correlation Between CCND1 Gene Polymorphism and Genetic Susceptibility to Gastric Cancer in Chinese Population


 Objective: The relationship between Cyclin D1 gene (CCND1) rs9344 polymorphism and susceptibility to gastric cancer (GC) is investigated.Methods: In a case-control study, we selected 577 cases of GC from The People's Hospital Affiliated to Jiangsu University in China along with 678 normal controls. Blood DNA was extracted and PCR amplified, gene polymorphism was determined using Snapshot method.Results: Analysis reveals significant difference in smoking between GC and control groups (P=0.006), however, not on polymorphism (P＞0.05). Conclusion: Smoking is associated with gastric cancer, whereas CCND1 rs9344 polymorphism does not implicate susceptibility of gastric cancer.


Introduction
Gastric cancer (GC) is one of the most common malignant tumors of the gastrointestinal tract and the second most common cause of cancer mortality worldwide. GC is a complex disease with multiple causes [1][2] , including helicobacter pylori infection, environmental dietary factors, and genetic factors. Single Nucleotide Polymorphism (SNP) [3] , as a type of genetic factors, is essential genetic information that affects susceptibility of human diseases, plays an important role in pathogenesis of GC. Cyclin D1 (CCND1) gene located in chromosome 11ql3 is the most important positive cell cycle regulator in the Cyclin family (Cyclin). A CCND1 forms a complex when binding to a cyclin-dependent kinase and stimulate cell transition from phase G1 to S [4] . CCND1 gene decrease cell cycle through gene ampli cation and protein overexpression, leading to independent proliferation of tumor cells [5][6] .
Rs9344 polymorphism has been proved to be associated with breast cancer [10] , rectal cancer [11] , lung cancer [12] , bladder cancer [13] , but there is few work on its relationship with GC. This paper aims to provide evidence for early screening, diagnosis and treatment of GC.

Materials And Methods
During May 2013 and June 2017, a hospital based investigation was conducted involving 1255 random samples (577 gastric cancer patients and 678 controls). The subject group consisted of 394 male and 183 female Han Chinese aged at 61.34±11.097 years old. The control group of 678 healthy people were selected from the same survey area without blood relationship. The difference was not statistically signi cant in occurrence distribution of age or gender between case and control groups (P 0.05). This research has been approved by the ethics review committee of Jiangsu University. A standardized training was conducted before study. Subjects who participated in this study were informed of signi cance.
DNA extraction, PCR ampli cation and Snapshot typing were used . Table 1 shows primer sequences of rs9344 locus were designed using software Primerpremier 5.0 software [14] . Extension primers were designed . The PCR reaction conditions were pre-denaturation at 95 o C for 3 minutes, denaturation at 94 o C for 30 seconds, annealing at 56 o C for 40 s, extension at 72 o C for 40 s, a total of 30 cycles, and the nal 72 o C extension is for 5 min. PCR ampli cation products were puri ed with ExoI and FastAP for the extension reaction, and ABI3730XL sequencer was used for sequencing and genotyping detection.

Results And General Features Of Samples
Results in Table 2 showed that there was no statistically signi cant difference between two groups' age (P=0.065). There was no statistically signi cant difference in gender frequency between the case and control groups (P=0.635). Smoking ratio in case group was 34.49% higher than control group 27.29%. Alcohol ratio was 21.49% in case lower than 23.30% in control group, alcohol consumption was not statistically signi cant difference between two groups (P=0.443).       Table 7 shows that strati ed analysis, we analyzed strati cation according to gender, age, smoking and drinking factors.
The results showed that differences were not statistically signi cant in heterozygous mutants, homozygous mutants, dominant models or recessive models.

Discussion
The early symptoms of GC are not obvious. Patients have often reached middle or late stages when diagnosed clearly, with a low 5 years survival rate of 30% [15,16] . Current treatments for GC includes surgery, chemotherapy, drug targeted therapy, etc. Finding speci c biomarkers is bene cial for early screening diagnosis, and clinical targeted drug therapy.
This project is based on the relationship between polymorphism in CCND1 gene and GC, with few similar studies currently reported. In 2010, Kuo et al [17] studied 358 GC patients and 358 healthy people and found that rs9344 locus polymorphism was associated with gastric cancer risk, and that subjects carrying variants AG and GG genotypes had lower risk than carrying AA genotypes. Rs9344 genotype maybe a useful biomarker for detection of early GC. Kuo et alalso investigated the interaction between rs9344 genotype and individual smoking status on GC risk and found that there was a synergistic effect between gene and smoking, which may increase the develop risk of GC. Another Yokoyama et al [18] suggested that there was no correlation between CCND1 rs9344 gene polymorphism and risk of GC in Japanese population in 2001. Our study suggest that there is no signi cant association between CCND1 rs9344 polymorphism and gastric cancer besides that smoking is a risk factor.
Alcohol drinking in this study was not related to gastric cancer, which is consistent with the results from Yokoyama et al, but differs from the research by Inoue M [19] . According to Inoue M research , drinking alcohol may be a potential risk factor for gastric cancer: the reason why drinking alcohol as risk factor may be that occurrence of gastric cancer is a slow process under joint action of multiple factors. Long-term heavy drinking can cause damage to gastric mucosa itself. On the other hand, alcohol intake in wine can stimulate gastric mucosa to reduce barrier function and increase gastric mucosa permeability, resulting in increased absorption of carcinogens. Another study by Tahara et al. [20][21] showed that CCND1 rs9344 polymorphism was associated with high methylation risk of CpG island promoter in gastric cancer, which was the rst report to show a potential association between CCND1 hypermethylation status in gastric cancer. A meta analysis [22] showed that analysis shows the relationship between site polymorphism and gastric cancer is different between Asian and Caucasian populations. Rs9344 polymorphism increases risk of GC in Caucasians but not in Asian.
The occurrence and development of gastric cancer is affected by various factors, in addition to genetic background, other factors that should be taken into consideration, may include environment, lifestyle, race, ethnicity, eating habits, genetic heterogeneity, sample size, etc. Therefore, the relationship between CCND1 gene and gastric cancer susceptibility needs further veri cation, larger samples, and elaborate design in the further .