| Objective: Mitochondria known as “power plant”, is an importantorganelle in eukaryotes, and is the center of energy metabolism in cells.Mitochondrial DNA (mtDNA) is the only genetic material outside the nucleusand it can complete the process of replication, transcription and translationindependently. mtDNA is believed to be more susceptible to damage thannuclear DNA (nDNA) due to exposure to a high oxygen environment directly,lacking of protective histones, and limited capacity for DNA repair. Thehuman mtDNA is a16569bp, closed circular, double-stranded molecule and itcontains coding region and non-coding region. The coding region encodes37genes, which composed by13protein genes, two ribosome RNAs and22tRNA. D-Loop region is the only non-coding region of mtDNA and is locatedin16026-577np, accounting for about6%of all mtDNA. This region isimportant for regulating both transcription and replication of mtDNA becauseit contains the important structures of replication origin of the heavy strandand transcription promoters of the light and heavy strand. It has been recentlyreported that D-Loop region mutations were associated with cancer risk andprognosis of many tumors, but there are few study on the relationship betweenpolymorphisms at D-Loop region of mtDNA and prognosis of gastric cancer.In this study, the relevance of polymorphisms at D-Loop region of mtDNAand prognosis of gastric cancer patients were analyzed, and the relationshipbetween clinical features and prognosis of gastric cancer patients wereassessed as well, which contribute to providing theoretical basis for areasonable treatment plan and prognosis evaluation of gastric cancer patients.Methods:1Sample collection and patients following-up:100gastric cancerpatients who were diagnosed by gastroscopy and histopathological method and underwent tumor resection in the Department of Gastrointestinal Surgeryat Fourth Hospital of Hebei Medical University from January2007toDecember2007were enrolled. Blood samples for experiment were collectedfrom patients before operation. The patients’ clinical characteristics wereaccurately recorded, and the3-years survival status of post-operation patientswere followed up by outpatient clinic, phones and letters. The follow-updeadline was January2011.2mtDNA extraction, amplification and sequencing: The mtDNA wasextracted with DNA purification kit from blood samples. The target gene ofqualified DNA samples were then amplified by PCR, the forward primer is5’-CCCCATGCTTACAAGCAAGT-3’, and the reverse primer is5’-GCTTTGAGGAGGTAAGCTAC-3’. The PCR products were confirmed byagarose gel electrophoresis and sequenced using the ABI Genetic Analyzer(3730XL). Polymorphisms were confirmed by repeating the analysis on bothstrands.3Statistical analysis: All analysis were performed by SPSS13.0software,P＜0.05was considered statistically significance. Kaplan-Meier method wasused to draw survival curves and its significance was tested by Log-Rankmethod. Factors related to the prognosis were analyzed by Cox regressionmodel. P-value, relative risk (RR) and95%confidence interval (CI) werecalculated.Results:1The univariate survival analysis showed that polymorphisms at D-Loopregion such as16519T/C,150C/T,489T/C and515-524(AC)n were associatedwith the prognosis of gastric cancer patients. The3-year survival rate of16519T genotype and C genotype were55.3%and33.9%, respectively.Patients of T genotype has longer survival time than C genotype patients(P=0.037). The3-year survival rate of150C genotype and T genotype were38.7%and58.7%, respectively. The length of survival of patients with Tgenotype was significantly longer than that of patients with C genotype(P=0.037). The3-year survival rate of489T genotype and C genotype were 56.3%and28.8%, respectively. The prognosis of patients with T genotypewas superior to that of patients with C genotype (P=0.002). The3-yearsurvival rate of515-524(AC)5genotypes and (AC)4genotypes were32.1%and54.5%, respectively. Patients of (AC)4genotypes has longer survival timethan C genotype patients (P=0.045).2The univariate survival analysis showed that clinical characteristics,such as gender, age, tumor location, degree of differentiation, intravasculartumor emboli were no relevance with the prognosis of gastric cancer patients.The3-year survival rates of stage Ⅰ+Ⅱ group and stage Ⅲ+Ⅳ group were78.1%and25.0%, respectively. Patients of stage Ⅰ+Ⅱ has longer survivaltime than stage Ⅲ+Ⅳ(P=0.000). As for tumor diameter, the3-year survivalrates of≤6cm group and＞6cm group were60.8%and22.4%, respectively.The length of survival of patients with diameter≤6cm was significantlylonger than that of patients with diameter＞6cm (P=0.000).3The factors which have significant difference in univariate analysiswere then analyzed by Cox regression model. It was showed that16519T/C,489T/C, tumor stage and tumor diameter were independent factors forprognosis of gastric cancer patients. The death risk of patients with Cgenotype was1.923times of T genotype at the16519allele. The death risk ofpatients with C genotype was2.109times of T genotype at the489allele. Themortality risk of patients with stage Ⅲ+Ⅳ was3.414times of stage Ⅰ+Ⅱ.The mortality risk of patients with diameter＞6cm was2.065times ofdiameter≤6cm.Conclusions:1The polymorphisms of16519T/C and489T/C of mtDNA wereindependent factors for prognosis of gastric cancer patients. The prognosis ofpatients with T genotype was superior to C genotype. The polymorphisms ofmtDNA is expected to become a new molecular marker to diagnosis of gastriccancer, and guide clinical treatment.2The tumor stage and tumor diameter were independent factors forprognosis of gastric cancer patients. The prognosis of patients with stage Ⅰ+ Ⅱ was superior to stage Ⅲ+Ⅳ. The length of survival of patients withdiameter≤6cm was significantly longer than that of patients with diameter＞6cm. |
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