| Gastric cancer is one of the most common cancers of China, which threatens the human’s life and health seriously. China now bears the greatest burden from gastric cancer of the world. Gastric cancer is the second most common malignancy and the third leading cause of cancer death of China. The tumorigenesis of gastric cancer is a complex process with multiple steps, and influenced by both environmental and genetic factors. Helicobacter Pylori infection, N-nitroso compounds and polycyclic aromatic hydrocarbons explosion, tobacoo use and alchol drinking have been demonstrated as the major risk factors in gastric cancer developmemt. In addition, the difference of inheritable background may also lead to different gastric cancer susceptibility. Cancer is disease with highly complexity and extensive heterogeneity. Although several factors related to gastric cancer have been found, more effort on mechanism researching is needed to find out the underlying cause of gastric cancer.The major role of mitochondria is to generate cellular adenosine triphosphate (ATP) through oxidative phosphorylation. Mitochondria are semiautonomous organelles within cells that play important roles in cellular energy metabolism, generation of reactive oxygen species (ROS), calcium homeostasis, and apoptosis. Mitochondria are semi-autonomously functioning organelles, which contain a resident genome, and replicate, translate and transcribe their own DNA. Human mtDNA is an extra-chromosomal circular, double-stranded, maternally-inherited DNA. MtDNA is more susceptible to oxidative damage and has a higher mutation rate compared with nuclear DNA due to a lack of protective histone proteins, limited DNA repair activities, and a high rate of generation of ROS in mitochondria. Somatic mutation and damage to mtDNA can result in impairment of the OXPHOS system and enhanced ROS production, which in turn accelerates the rate of DNA mutation. This scenario has been proposed to be involved in the initiation of carcinogenesis.Mitochondrial DNA copy number (mtCN) in each cell is usually according to the energy needed to a cell, in order to maintain normal physiological function. Some reports have hypothesized that the variations in the mtDNA copy number reflect the results of gene-environment interactions between genetic factors and the levels of oxidative stress (an imbalance between ROS production and the antioxidant capacity), caused by a variety of endogenous and exogenous factors, such as hormones, age, dietary and environmental oxidants/antioxidants, and reaction to oxidative damage, all of which are thought to be risk factors for various types of cancer. It is suggested that mtDNA copy number may be a kind of biomarkers to predict cancer risk. Over the past few years, an increasing number of epidemiologic studies have evaluated the cancer risk by measuring the mtCN in surrogate tissues, mainly in peripheral blood leukocytes. However, results from previous studies have been conflicting. Only two recent studies with relative small sample size, reported significant associations between mtCN and gastric cancer risk. Therefore, further studies based enough samples are warranted to clearly illustrate the correlation between mtDNA copy number and gastric cancer risk.The heterogeneity of mtDNA copy number exists widely between different individuals. It was reported that some nuclear DNA encoding genes involved in regulation of mtDNA copy number. Hence, mtDNA copy number not only affected by environmental factors, but also influenced by genetic factors. It is estimated that the heritability of mtDNA copy number varying between 32.9% and 65%. Genome-wide association studies (GWAS) is the best strategy to appraise genetic markers comprehensively. However, there is no genome-wide association studies of mtDNA copy number based on whole genome.Therefore, we firstly conducted a case-control study with a relative large sample size to investigate the relationship between mtDNA copy number and gastric cancer risk. Furthermore, we conducted a genome-wide association study to explore the relationship between mtDNA copy number and genetic variants and futher evaluate the effect of mtDNA copy number related genetic variants on gastric cancer susceptibility.Part I:A case-control study on the association between mtDNA copy number and gastric cancer riskMitochondrial dysfunction and alterations of mitochondrial biogenesis have been documented in age-related diseases, including various types of cancer. The significant associations between mtCN and gastric cancer risk have been reported in several epidemiology studies. However, the sample sizes of these studies were small and results were inconsistent. Sun et al conducted a study in 132 gastric cancer cases and 125 controls of a Hispanic population and reported a significant association between low mtCN and increased gastric cancer risk, when individuals were dichotomized into high and low mtCN groups based on the median mtCN value in the controls. In the other study with 162 gastric cancer cases and 299 controls of a Chinese population, Liao et al found that there is no association between leukocyte mtCN and risk of gastric cancer.Therefore, we conducted a case-control study with relatively large sample size to investigate the relationship between mtCN and gastric cancer risk.984 gastric cancer cases were recruited from hospitals in Jiangsu province.984 cancer-free controls were randomly selected from a community-based screening program for non-infectious diseases, and were frequency-matched to cases based on age and sex. We use quantitative polymerase chain reaction (PCR) method to measure mtCN. In brief, mtCN was calculated based on the copy number measurement of mitochondrial gene (NADH dehydrogenase, subunit 1, ND1) and nuclear gene (hemoglobin subunit p, HGB) expressed as the ratio of ND1 and HGB threshold cycle numbers ratio in individual samples. All samples were run in duplicate and the mean of two measurements was used in the statistical analyses. Technicians were blinded to the case-control status. Logistic regression was used to analyze the gastric cancer risk of subjects in different mtCN group, and compute the OR and 95%CI.As a result, the mtCN in cases (median:6.53, inter-quartile range [IQR]: 2.61-20.84) was higher than that in controls (4.12,2.13-13.95, P<0.001). Log-transformed mtDNA copy numbers (logmtCN) were divided into three categories (low, median, and high) based on the tertiles distribution of logmtCN in controls to examine the association between mtCN and gastric cancer risk.Compared to the low-logmtCN group, a significant dose-response effect (Ptrend= 1.67x10-6) was observed between increased risk of gastric cancer and increasing mtCN, with ORs 1.28 (95%CI=1.02-1.62) for the median log_mtCN group, and 1.73 (95% CI=1.38-2.17) for the high-log_mtCN group.These results collectively suggest that high mtCN was associated with an increased risk of gastric cancer. Further studies based on prospective design and enough sample size may facilitate to confirm our findings. This study provided valuable clues for better understanding the underlying contribution of mtDNA copy number to gastric carcinogenesis. Our results suggested that mtDNA copy number may be a kind of biomarkers to predict cancer risk, and provided theoretical and technical support to screening and the prevention and control for gastric cancer in high-risk population.Part II:Genome-wide association study for mtDNA copy numberGenetic factors may be one of the causes of differences between mtDNA copy number in individuals, and the, which impact mtDNA copy number, is unclear. Genome-wide association study (GWAS) have been widely used in association studies between phenotype and genetic variants. Therefore, we can perform GWAS identified relevant genetic loci of impacting mtDNA copy number.We performed an mtDNA copy number GWAS analysis in a Chinese cohort based on Affymetrix GeneChip Human SNP Array 6.0 Chip. The GWAS comprised 984 individuals from Changzhou community-based screening program for non-infectious diseases, and these 984 individuals also measured the mtDNA copy number according to a quantitative real-time polymerase chain reaction (PCR)-based method. Generalized linear models were used to conduct tests for correlation between mtCN and genetic variants. Logistic regression was used to estimate the OR and 95%CI for gastric cancer risk in each genetic variant.As a result, rs28648793 located in 16q22.2 was significantly associated with mtDNA copy number in our population. rs137876970 (9q32), rs10481674 (9q32), rs138666129 (8q23.3), rs1860742 (7q36.1), rs6467800 (7q34), rs6938637 (6p24.3), rs 1935171 (1q32.1) were potential significantly associated with the mtDNA copy number in the GWAS analysis (5×10-8<P<10-5). Then, we evaluated the association between these mtDNA copy number related variants and gastric cancer risk. We found rs10481674-C, located in 9q32 region, had a significantly associated with increasing mtDNA copy number (P=0.804, P=3.58×10-6) and exhibited a significant association with gastric cancer risk (OR=1.51, P=0.015). However, we found other genetic loci were no significantly associated with gastric cancer risk.In this study, we sysmatically evaluated the relationship between mtDNA copy number, genetic variants and gastric cancer risk for the first time. And we found genetic variants located in 16q22.2 and other seven regions may affect the mtDNA copy number. Furthermore, we found rs10481674 located in 9q32 had a significantly associated with increasing mtDNA copy numberand exhibited a significant association with gastric cancer risk. Our not only provided valuable clues for better understanding the genetic regulating mechanism of mtDNA copy number, but also, our methodology and data resources may as an important reference for further studies. |
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