| Objective: Gastric cancer is the first leading cause of cancer-related mortality and remains a significant cancer burden currently in China due to the high metastasis, poor prognosis and delay of diagnosis, so it becomes one of the key issues in cancer prevention and control strategy. Recently, the connection between Oxidative damaged and carcinogenesis of gastric cancer has been the study hotspot. Oxidative damage effects the function and the structure of macromolecules including proteins, lipids and DNA, is therefore an important contributor to gene mutation, cancer and human ageing. Mitochondrial DNA(mt DNA) is believed to be more susceptible to damage than nuclear DNA due to exposure to a high oxygen environment directly, limited capacity for DNA repair and lacking of protective histones. The displacement loop(D-Loop), which important for regulating both transcription and replication of mt DNA, becomes a mutation hotspot of mt DNA. It was reported that accumulation of D-loop sequence alterations may contribute to carcinogenesis of gastric cancer. The 73 G, 523-524 Del and 16519 C genotypes were significantly associated with an increased risk for gastric cancer, whereas the 309 C insert and 16362 C genotypes might be associated with resistance to gastric cancer. Therefore, it is likely that the polymorphisms in the D-Loop may affect the energy production and oxidative stress, which cause the occurrence of gastric cancer. The 8-hydroxy-2’-deoxyguanosine(8-OHd G), which is one of the main mutagenic modifications of DNA by oxidative stress, induced G-C to T-A transformation in daughter DNA strands. It has been confirmed that the expression of 8-OHd G in patients with nasopharyngeal carcinoma, lung cancer, Hepatocellular carcinoma, ovarian carcinomas, colorectal cancer and so on was higher than normal, so 8-OHd G could be a useful marker for assessing the progress and prognosis of malignant tumor. In this study, the expression of 8-OHd G was measured by immunohistochemistry method in gastric cancer samples, the relationship between expression of 8-OHd G and the single nucleotide polymorphisms(SNPs) that associated with the risk of gastric cancer in D-Loop region of mt DNA was assessed, and the relevance of expression level and clinical features and prognosis of gastric cancer patients was analyzed as well, which contributed to exploring the process of gastric cancer and making diagnostic and prognosis evaluation earlier.Methods:1 Sample collection and patients following-up: 131 gastric cancer patients who were diagnosed by gastroscopy and histopathological method and underwent tumor resection in the Department of Gastrointestinal Surgery at Fourth Hospital of Hebei Medical University from March 2007 to August 2008 were enrolled. All of the gastric cancer patients didn’t have chemotherapy, radiation and other treatment before surgery, the pathology results of patients were confirmed in the Department of Pathology at Fourth Hospital of Hebei Medical University by more than two experienced physicians. The patients’ clinical characteristics were accurately recorded, such as gender, age, degree of differentiation, tumor thrombus, TNM classification, diameter of the tumor and so on. The 3-years survival status of post-operation patients were followed up by outpatient clinic, letters and phones. The follow-up deadline was August 2011. Tissue samples for experiment were collected from the Department of Pathology at Fourth Hospital of Hebei Medical University and all the samples were supervised and approved by the hospital’s Human Tissue Research Committee.2 DNA amplification and sequencing: The target gene of qualified DNA samples were amplified by PCR. The forward primer was 5’-CCCCATGCTTACAAGCAAGT-3’ and the reverse primer was 5’-CATGC- TGGTGTGACACAGTC-3’. The PCR products were confirmed by agarose gel electrophoresis and sequenced using the ABI Genetic Analyzer(3730XL). Polymorphisms were confirmed by repeating the analysis on both strands.3 Measurement of 8-OHd G in gastric cancer tissues: tissue(4 μm) of gastric cancer were prepared for Immunohistochemistry analysis, and then Dewaxing with xylene and dehydrating in absolute ethanol. Tissue sections were incubated for 2 days at 4?C with anti-8-OHd G antibody(dilution 1:100) and then reacted with biotinylated secondary anti-mouse Ig G antibody for 60 minutes at room temperature. Streptavidin was added and the color was developed with 3,3’-diaminobenzidine(DAB).The level of 8-OHd G was measured as described. Two pathologists counted the number of 8-Ohd G-stained in 10 random fields(magnification, x400). We calculated the percentage of positively stained cells, which was termed the 8-OHd G label index(LI), in each field and graded them as follows: low, LI ≤50%; high, LI >50%.4 Statistical analysis: The survival data were analyzed by the Kaplan-Meier method, differences were determined by the log-rank test. Differences in clinical characteristics and the SNPs of D-Loop region between the high and low LI groups were compared with the χ2 test. P≤0.05 was considered to be statistically significant and all calculations were performed using SPSS 13.0 software.Results:1 It had been found that the risk of gastric cancer was associated with the SNPs of D-Loop. Differences in SNPs of D-Loop region between the high and low LI groups were compared with the χ2 test, which associated with the risk of gastric cancer, such as 523-524AC/del, 16362T/C, 16519C/T and 309C/C insert. It showed that the expression of 8-OHd G in 16519 C was higher than in 16519T(P=0.017), there was no significant different in the other regions(P>0.05).2 In 131 Gastric cancer patients, the Immunohistochemistry of 8-OHd G observed in Gastric cancer tissues occurred mostly in the nuclei. The relationship between the level of 8-OHd G and the 3-year survival rate of patients was analyzed also, and it was found that the 3-year survival rate was 46.81% for the high group and 46.43% for the low group. The log-rank test did not reveal a markedly significant difference(P>0.05) between the low and high LI groups demonstrating the uselessness of the oxidative DNA damage marker 8-OHd G in predicting the prognosis of gastric cancer.3 The correlation of clinical characteristics and 8-OHd G levels was evaluated in gastric cancer patients using the χ2 test. It showed that age, gender, TNM classification, diameter of tumor, tumor location, degree of differentiation, intravascular tumor emboli were not correlated with 8-OHd G levels(P>0.05).Conclusions:1 Previous study had found that 16519 C genotype increased the risk of gastric cancer, and the oxidative damage degree of 16519 C genotype was significantly higher than 16519 T genotype. It is likely that the polymorphisms in the D-Loop may affect the energy production and oxidative stress, which cause the occurrence of gastric cancer.2 The level of 8-OHd G alone should not be an independent prognostic factor for judging the prognosis of gastric cancer.3 The clinical characteristics such as age, gender, TNM classification, diameter of tumor, tumor location, degree of differentiation, intravascular tumor emboli were not correlated with 8-OHd G levels in gastric cancer patients. |
PDF Full Text Request