The Relevance Of Sequence Polymorphisms In The COX Region Of Mitochondrial DNA And Occurrence Of Gastric Cancer

Objective:Gastric cancer,one of the common malignant tumors in the world,is a serious threat to human health because of the high morbidity and poor prognosis.In recent years,more and more attention has been paid to the research on the relationship between mitochondrial DNA mutation and single nucleotide polymorphism(SNPs)and tumorigenesis.Mitochondrial DNA(mtDNA)is the only genetic material outside the nucleus,encoding the polypeptide involved in oxidative phosphorylation and the production of ATP.MtDNA is more prone to oxidative damage and mutation because of the lack of protective histone and limited capacity for DNA repair system.MtDNA consists of two parts: the coding region and the non-coding region.The mt DNA coding region contains 13 kinds of genes coding polypeptide,two rRNA genes and 22 tRNA genes while the D-Loop region is the only non-coding region of the gene.As one of the genes in the mtDNA coding region,COX gene is responsible for encoding three important subunits of cytochrome oxidase(cytochrome oxidase,COX).Cytochrome oxidase is a key enzyme in the transmission of mitochondrial respiratory chain.The mutation and polymorphism of COX gene may be associated with tumorigenesis.In this study,the entire section of COX gene of gastric cancer patients and heathy controls were amplified and sequenced to look for the SNPs associated with gastric cancer and the relationship between the gastric cancer risk-associated SNPs and clinical features was analyzed to explore the potential role of mtDNA polymorphisms in occurrence and progression of gastric cancer and to provide new ideas for the early diagnosis and treatment of gastric cancer.Methods:1 Research objects and sample collection:100 gastric cancer patients who underwent curative resection in the department of general surgery at the Fourth Hospital of Hebei Medical University during the period from May2007 to August 2008 were enrolled.All patients were diagnosed as gastric cancer by endoscopy and histopathology before operation.100 healthy persons who were examined at the Fourth Hospital of Hebei Medical University during the period from September 2013 to June 2014 were enrolled.After the access to informed consent,peripheral venous blood samples were collected from gastric cancer patients and normal people,respectively.Both the clinical characteristics of the gastric cancer patients and the physical examination data of the control group were observed and recorded.2 Mitochondrial DNA extraction:The genomic DNA was extracted from venous blood samples of patients with gastric cancer and persons in the healthy controls with a Wizard Genomic DNA extraction kit and it was kept in the refrigerator after the quantitative analysis of mtDNA.3 Amplification and sequencing of mitochondrial DNA COX gene:The COX gene,including COX I,COX II and COX ?,was divided into 9 small gene fragments according to the length of 600 bps.The upstream and downstream primers for each segment of the small gene fragment were designed and then the gene fragment was amplified by PCR and purified by agarose gel electrophoresis.The gel imaging system(Image)was used to test whether the DNA bands were amplified and if amplified well,they would be sequenced in Biotechnology(Beijing)Company.4 Statistical analysis:?2 test was used to analyze enumeration data.For the measurement data,t test was used when the number of cases was less than 30 and U test was used when the number of cases was greater than or equal to 30.All of the statistical analysis was performed with the SPSS 21.0 software.A P-value <0.05 was considered statistically significant.Results:1 There was no significant difference in age and gender for clinical characteristics between gastric cancer patients and heathy controls(P>0.05).2 In the gastric cancer group and control group,28 cases of DNA were randomly selected to be finished the amplification and sequencing of the whole COX gene.It was shown in the study there were 9 high frequency polymorphism loci(frequency >5%),including 6392T/C,6455C/T,6962G/A,7196C/A,7853G/A,9540T/C,9548G/A,9824T/C and 9950T/C.There were critical differences of the SNPs 9540 T/C frequency distribution between gastric cancer group and the control group(?2 test =3.541,P=0.06).The 9548G/A frequency distribution between gastric cancer group and control group(?2=1.409,P=0.235)may also be statistically significant.The distribution of SNPs 9540 T/C and 9548 G/A may be associated with the risk of gastric cancer.3 After the number of cases of 9540 T/C and 9548 G/A in COX? gene increased,the SNPs 9540 T/C distribution frequency was statistically significant(?2=4.504,P=0.034).It was also found that the distribution frequency of SNPs 9548G/A was statistically different between the case group and the control group(?2=4.188,P=0.041).4 We analyzed the relationship between the gastric cancer risk-associated SNPs and clinical features of gastric cancer patients.The result showed that there was no association of 9540T/C,9548G/A SNPs with clinical features,including age,gender,tumor size,extent of differentiation,tumor stage,intravascular tumor thrombus and nerve invaded.Conclusions:1 The SNPs for 9540 T/C and 9548G/A were associated with the risk of gastric cancer.The 9540 C genotype might reduce the risk of gastric cancer,while 9548 A genotype might be associated with an increased risk.2 There was no association between the genotype distribution of 9540T/C and 9545 A/G with clinical characteristics in gastric cancer patients.