| Objective: Gastric cancer is one of the most frequent cancers in theworld and threatens people’s health due to the high mortality rate. Recently, itwas noticed that the mitochondrial DNA (mtDNA) mutation and singlenucleotide polymorphisms (SNPs) were associated with the occurrence ofcancers. The human mtDNA, as the only genetic material of extranuclear, is a16569bp, circular and double-stranded DNA coding for13polypeptidesinvolved in respiratory chain complexes, as well as22transfer RNAs and2ribosomal RNAs involved in protein synthesis in mitochondria. Thedisplacement loop (D-Loop) is the only non-coding mtDNA region which isimportant for regulating both replication and expression of mitochondrialgenome because it contains the leading-strand origin of replication and themain promoter for transcription. It has been reported that the D-Loop region isa hotspot for mutations and polymorphisms in different types of cancers,including gastric cancer. SNPs is the polymorphisms induced by singlenucleotide variation in the DNA sequence, which has been widely applied toresearch susceptible genes of multigenic disease. In this study, the D-Loopregion of mtDNA was sequenced on a series of145gastric cancer patients and159heathy controls, the relationship between the gastric cancerrisk-associated SNPs and clinical features was evaluated, and the potential roleof mtDNA polymorphisms in occurrence and progression of tumors wasexplored, which would be helpful to detect valuable markers for gastric cancerrisk evaluation and make therapeutic decisions earlier.Methods:1Object of study and sample collection: Blood samples were collectedfrom145gastric cancer patients who underwent gastric cancer resection in thedepartment of general surgery at the Fourth Hospital of Hebei University from 2007to2008. All patients were diagnosed histopathologically beforeoperation. Blood samples were also collected from159healthy controlswithout a family history of gastric cancer who were checked-up in the samehospital. Informed consents were obtained from all participants prior toenrollment and all the samples were collected according to the Human TissueResearch Committee at the hospital.2DNA extraction, amplification and sequencing: The genomic DNA wasimmediately extracted with the Wizard Genomic DNA extraction kit. PCRwas run with a PCR Master Mix Kit (Promega). The forward primer was5’-CCCCATGCTTACAAGCAAGT-3’ and the reverse primer was5’-CATGC-TGGTGTGACACAGTC-3’. Cycle sequencing was performed with the ABIPRISM BigDye Terminator v3.1Cycle Sequencing Kit (Life Technologies,Carlsbad, CA, USA), and the products were then read with the ABI PRISM3100Genetic Analyzer (Life Technologies). The products were confirmed byagarose gel electrophoresis and sequencing.3Statistical analysis: Student’s u-test was used to analyze measurementdata, and χ2test was used to analyze enumeration data. The magnitude ofassociation was estimated as odds ratio (OR), using95%confidence intervals(95%CI). All of the statistical analysis was carried out with the SPSS19.0software. A P-value <0.05was considered statistically significant.Results:1As for clinical characteristics, there was no statistical difference in ageand gender between gastric cancer patients and heathy controls (P＞0.05).2One hundred and forty five gastric cancer patients and159healthycontrols were enrolled in this study. SNPs were detected in152sites withinthe982-bp mitochondria D-Loop region from the blood samples of the healthycontrols and gastric cancer patients, and the frequency of the mtDNAalterations was15.5%. Among these, seven SNPs without reporting in theMitomap database were classified as new polymorphisms. The SNPs withsample size over five in either gastric cancer patients or controls were used forcancer risk analysis, a total of27SNPs were selected. 3When individual SNPs were analyzed comparing gastric cancer patientswith the healthy controls, a statistically significant difference of distributionfrequency was observed for73G/A,309C/C insert,523-524AC/del,16362T/Cand16519C/T alleles. The frequency of genotype73G was100.0%in gastriccancer patients and56.0%in controls. The allele frequency of73G in gastriccancer group was higher than that in control group (P=0.000). The insertionsof one or two base pairs in the309repeat were detected in64of145gastriccancer patients (44.1%) and101of159healthy controls (63.5%). The allelefrequency of309C insert in gastric cancer group was lower than that in controlgroup (P=0.001). The AC deletion at523-524sites was detected in71of145patients (49.0%) and57of159controls (35.8%). The allele frequency of523-524Del was higher in gastric cancer group compared with control group(P=0.021). In addition, the frequency of genotype16362C was29.0%ingastric cancer patients and47.2%in controls. The allele frequency of16362Cin gastric cancer group was lower than that in control group (P=0.001). Theallele frequency of16519C in gastric cancer group (62.1%) was higher thanthat in control group (49.1%)(P=0.023).4The relationship between the gastric cancer risk-associated SNPs andclinical features of gastric cancer patients was then analyzed. The resultshowed the allele frequency of16362C in≤60years group (38.0%) washigher than that in>60years group (20.3%)(P=0.018), while the allelefrequency of16519C tended to be higher in patients without intravasculartumor thrombus (68.6%) than in patients with intravascular tumor thrombus(45.0%)(P=0.009). There was no association of other gastric cancerrisk-associated SNPs with clinical features, including gender, age, tumor size,tumor site, stage, extent of differentiation and intravascular tumor thrombus.Conclusions:1The D-Loop region of mtDNA was a hotspot for polymorphisms ingastric cancer. Most of the mtDNA variants were single base transition,insertion and deletion, and mainly located in the hypervariable segment one(HV1) and hypervariable segment two (HV2). 2The mtDNA D-Loop polymorphisms may be associated with thetumorigenesis and progression of gastric cancer. The73G,523-524Del and16519C genotypes were significantly associated with an increased risk forgastric cancer, whereas the309C insert and16362C genotypes might beassociated with resistance to gastric cancer.3There was no correlations between D-Loop polymorphisms and clinicalfeatures of gastric cancer patients, which meant that the D-Looppolymorphisms may potentially play a role in the early stage of tumorprogression and be useful biomarkers for predicting gastric cancer risk. |
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