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RESEARCH ARTICLE

High mitochondrial DNA copy number was associated with an increased gastric cancer risk in a Chinese population† Xun Zhu1, 2, 3†, Yingying Mao1, 4†, Tongtong Huang1†, Caiwang Yan1, Fei Yu1, Jiangbo Du1, Juncheng Dai1, Hongxia Ma1, Guangfu Jin1,2*

1

Department of Epidemiology and Biostatistics, School of Public Health, Nanjing

Medical University, Nanjing 211166, China 2

Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Collaborative

Innovation Center for Cancer Medicine, Nanjing Medical University, Nanjing 211166, China. 3

Wuxi Center for Disease Control and Prevention, Wuxi, 214023, China

4

Department of Epidemiology and Biostatistics, School of Basic Medical Sciences,

Zhejiang Chinese Medical University, Hangzhou, 310053, China

†

Zhu X, Mao Y, and Huang T contributed equally to this work.

*

Correspondence to: Guangfu Jin, Department of Epidemiology and Biostatistics,

School of Public Health, Nanjing Medical University, Nanjing 211166, China, Tel: +86-25-8686-8437, Fax: +86-25-8686-8499, E-mail: [email protected].

†

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/mc.22703] Additional Supporting Information may be found in the online version of this article. Received 24 January 2017; Revised 6 June 2017; Accepted 7 July 2017 Molecular Carcinogenesis This article is protected by copyright. All rights reserved DOI 10.1002/mc.22703 This article is protected by copyright. All rights reserved

 

Abstract Mitochondrial DNA (mtDNA) copy number (mtCN) may be a potential biomarker in relation to cancer risk. However, the role of mtCN in gastric cancer remains uncertain. We examined the association between peripheral blood leukocytes mtCN level and gastric cancer risk in a case-control study including 984 gastric cancer cases and 984 controls. We measured relative mtCN level by real-time quantitative PCR-based assay, and used logistic regression models to assess the association between mtCN and risk of gastric cancer. The mtCN level in gastric cancer cases was significantly higher than that in controls (median value: 6.53 versus 4.12, P=1.79×10-5). Compared with those with low mtCN, the risk for gastric cancer was 1.29 (95% confidence interval (CI) = 1.02-1.63) in the median group and 1.74 (95%CI = 1.39-2.18) in the high mtCN group (P for trend = 1.51×10-6). Because relative telomere length (RTL) has been associated with gastric cancer risk in our previous study, we also evaluated the combined effects of mtCN and RTL on gastric cancer risk. Multivariable regression model revealed that the effects of mtCN and RTL were independent on gastric cancer risk. Compared with those in the lowest risk group by combining mtCN and RTL, the odds ratio for gastric cancer was 4.30 (95% CI = 2.79-6.63) in the highest risk group. Our results suggest that mtDNA may be implicated in gastric carcinogenesis and mtCN as well as RTL may serve as joint susceptible biomarkers for gastric cancer. This article is protected by copyright. All rights reserved

Keywords: mtDNA copy number; gastric cancer risk; association; telomere length.

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Abbreviations CI, confidence interval; Ct, threshold cycle; HGB, human globulin; IQR, interquartile range; mtCN, mitochondrial DNA copy number; mtDNA, mitochondrial DNA; MT-ND1, mitochondrially Encoded NADH:Ubiquinone Oxidoreductase Core Subunit 1; OR, odds ratio; RTL, relative telomere length; SD, standard deviation; TEL1, Telomere Maintenance 1

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Introduction Gastric cancer is the fifth most common malignancy worldwide, accounting for an estimate of 952,000 new cases annually [1]. Gastric cancer is separated anatomically into non-cardia gastric cancers, of which the incidence has been declining, and cardia gastric cancer, of which the incidence has been increasing [2]. Gastric cancer arises from the complex interplay between genetic and environmental factors, and Helicobacter pylori infection is considered as the predominant risk factor for gastric cancer [3]. Other modifiable risk factors included alcohol drinking, obesity, salty foods and processed meat intake [4, 5]. The incidence of gastric cancer varies considerably by geographical regions, with approximately half of all cases occurring in China [1]. According to the Chinese National Office for Cancer Prevention and Control, gastric cancer was the second most common cancer in China, with an estimate of over 679,000 new cases in 2015 [6]. The main factors that appear to be responsible for high gastric cancer rates in China include chronic infection with Helicobacter pylori, which is very common in China [7, 8], and lifestyle-related factors such as smoking, alcohol drinking, and unhealthy diet, especially foods preserved with nitrates and nitrites [4, 5]. Because a lack of symptoms in the early course of the disease, gastric cancer is often diagnosed at an advanced stage which resulted in poor prognosis. Substantial efforts have been made to develop bio-markers to identify individuals at high risk for gastric cancer; however, no efficacious screening strategies are available to date. Mitochondria are essential organelles in the cytoplasm of eukaryotic cells.

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Mitochondria possess their own genome and play pivotal roles in numerous biological processes such as ATP production, reactive oxygen species production, iron and calcium homeostasis, and autophagic cell death and apoptosis [9, 10]. The human mitochondrial DNA (mtDNA) is a 16569 bp circular double-stranded molecule which encodes 13 polypeptide subunits of the respiratory chain apparatus [11]. In cells under normal physiological conditions, the amount of mtDNA keeps relatively stable [12]. Recently, alterations in mtDNA copy number (mtCN) have been described in many different types of cancer, and may serve as a potential susceptible/diagnostic biomarker associated with cancer risk. For instance, several studies reported that low mtCN was associated with increased risk of esophageal adenocarcinoma [13], renal cell carcinoma [14, 15], oral squamous cell carcinoma [16] and nasopharyngeal carcinoma [17], while others found high mtCN in cancer patients, including colorectal cancer [18, 19], prostate cancer [20], renal cell carcinoma [21], breast cancer [22, 23], lung cancer [24], non-Hodgkin lymphoma [25], and glioma [26]. However, the association between mtCN and gastric cancer remains conflicting. Pilot data from a recent retrospective case-control study in Hispanic Americans showed that low mtCN was associated with increased risk of gastric cancer [27]. Yet, a prospective study of Chinese women found no association between leukocyte mtCN and risk of gastric cancer [28]. In the current study, we tested for association between peripheral leukocytes mtCN and gastric cancer risk in 984 gastric cancer patients and 984 age and sex frequency-matching controls. Since relative telomere length (RTL), which was

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considered crucial in maintaining chromosomal stability and integrity, was associated with risk of gastric cancer in our previous study [29], we also examined the correlation between mtCN and RTL as well as their joint effect on gastric cancer risk. Materials and methods Study population The study was approved by the Institutional Review Board of Nanjing Medical University, and written informed consent was obtained from each study subject. Details of the study population and recruitment protocols have been described previously [29]. Briefly, patients with gastric cancer were recruited from hospitals in Jiangsu province in Eastern China between 2004 and 2010. Eligible cases were incident and histopathologically confirmed gastric cancer (International Classification of Diseases for Oncology, C160-C169), had no previous history of cancer, and were not treated with radiotherapy or chemotherapy before enrollment. Gastric cancer cases were classified into cardia and non-cardia cancer according to location of the tumor. Cardia cancer was defined as cancer occurring at the top part of the stomach closest to the esophagus, and noncardia cancer was defined as cancer occurring in all other areas of the stomach. At the same period, healthy controls were recruited from a community-based screening program for non-infectious diseases in Jiangsu province and frequency-matched to cases by sex and age group (5-year interval). All study subjects were of Chinese Han ancestry.

Questionnaire data collection

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Every study subject was face-to-face interviewed by a trained interviewer, who administered a standard questionnaire including demographic data and lifestyle related factors such as cigarette smoking and alcohol drinking. Individuals who smoked at least one cigarette a day for more than one year were defined as smokers, and those who drank twice or more per week for at least one year were considered as drinkers. After interview, 5 ml peripheral blood sample was collected from each participant. Because of continuous experiments, 152 cases and 28 controls with DNA concentration less than 5ng/μl were excluded from PCR-based analysis for mtCN. As a result, a total of 984 cases (418 cardia cancer and 566 non-cardia cancer) and 984 controls were included in the final analyses. The descriptive characteristics of the study population are summarized in Supplementary Table 1. Gastric cancer cases (42.78%) in our study were more likely to be drinkers compared with controls (37.60%), but no other demographic or lifestyle characteristics differed significantly between cases and controls.

Determination of mtDNA copy number Total DNA was extracted from peripheral blood leukocytes by proteinase K digestion followed by phenol–chloroform extraction and ethanol precipitation. Relative mtCN was measured using quantitative real-time PCR-based assay in a high-throughput 384-well format with 7900HT Real Time PCR system (Applied Biosystems) as previously described [30, 31]. Briefly, two primer pairs were used, with one pair designed to amplify the mitochondrial subunit ND1 gene (MT-ND1), and another

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designed to amplify the single-copy nuclear gene human globulin (HGB). The primer sequences for ND1 were F, 5'-CCCTAAAACCCGCCACATCT-3' and R, 5'-GAGCGATGGTGAGAGCTAAGGT-3', and the primer sequences for HGB were F, 5'-GAAGAGCCAAGGACAGGTAC-3' and R, 5'-CAACTTCATCCACGTTCACC-3'. Each reaction contained 10 μl SYBR® Green PCR Master Mix (Applied Biosystems) with a final DNA concentration of 5ng/μl. The thermal cycling profile proceeded as follows: 50°C for 2 minutes, then 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute (for MT-ND1) or 56 °C for 1 minute (for HGB). Relative mtCN was calculated as the ratio of MT-ND1 to HGB copy number using standard curves. For each 384-well plate, DNA samples from five healthy controls were equally pooled as the reference and then serially diluted 1:2 to produce a six-point standard curve from 0.625 to 20ng/μl. The purpose of the standard curve is to assess interpolate variation in PCR efficiency. The R2 coefficient of determination value was ≥ 0.99 for each reaction. Samples with threshold cycle (Ct) values that fell outside the range defined by the standard curves were rerun at a different concentration to ensure that they were amplified within the linear range. qPCR for MT-ND1 and HGB was performed on separate plates, and laboratory personnel were blinded with regard to case-control status. Each sample was assayed in triplicates in the same run and the mean of the three measurements was used in the statistical analyses. The ratio of MT-ND1 to HGB (-dCt) for each sample was calculated by subtracting the average HGB Ct value from the average MT-ND1 Ct value. The

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relative ratio of MT-ND1 to HGB (-ddCt) was calculated by subtracting the ratio of MT-ND1 to HGB of the calibrator DNA from the ratio of each sample. Relative mtCN was calculated using the formula 2×2-ddCt [32]. Quality control samples were interspersed throughout assays, and the average inter-plate and the intra-plate variations were 9.08% and 7.71%, respectively.

Measurement of relative telomere length The measurement of relative telomere length in peripheral leukocytes was previously described [32]. Briefly, RTL was determined as the ratio of telomere repeat copy number (T) to single-copy gene copy number (S). The reference DNA pool from five healthy controls was used to generate a standard curve with concentrations ranging from 0.25 to 8ng/µl, and linearity (r2 > 0.99) over this range of input DNAs was observed. The telomere primers were TEL1, 5’-GGTTTTTGA[GGGTGA]4GGGT-3’ and TEL2, 5’-TCCCGACTAT[CCCTAT]4CCCTA-3’. The single-copy gene (36B4) primers were 36B4u, 5’-CAGCAAGTGGGAAGGTGTAATCC-3’ and 36B4d, 5’-CCCATTCTATCATCAACGGGTACAA-3’. Each sample was run in duplicate and the mean of the two measurements was used in the statistical analyses. RTL was calculated according to Cawthon’s formula: 2-ΔΔCt [32]. Similar quality control procedures were applied. In general, equal cases and controls were assayed on each 384-well plate, and technicians were blinded to the case-control status.

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Statistical Analysis All statistical analyses were performed using Stata software package version 9.2 (Stata Corporation, College Station, TX), unless otherwise noted. Student t-test and Pearson χ2 test were used to compare the differences in the distribution of selected characteristics between cases and controls. Rank-sum test was used to examine the differences of mtDNA copy number and relative telomere length between cases and controls. Spearman rank correlation test was used to examine the association of mtCN with RTL. We categorized subjects into three mtCN groups (low, median, and high) based on the relative mtCN among controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated for the association of mtCN with gastric cancer risk using unconditional multivariable logistic regression models with adjustment for age, sex, cigarette smoking and alcohol drinking status. Statistical significance of interaction of mtCN and RTL was examined by adding an interaction term in the multivariable logistic regression model. Also, we used Spearman rank correlation test to evaluate the relationship of mtCN and RTL. All analyses were two sided, and p values less than 0.05 were considered statistically significant.

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Results A total of 984 gastric cancer patients and 984 controls were included in the analyses. Among controls, mtDNA copy number was positively correlated with age (r=0.085, P = 0.008 for Spearman correlation test), but was not associated with sex (P = 0.834), cigarette smoking (P = 0.758), or alcohol drinking status (P = 0.942) (Table 1). The median mtCN value was higher in non-cardia gastric cancer patients than that in cardia cancer cases (6.85 versus 5.82), but the difference was not statistically significant (P = 0.119) (Table 1). The median leukocyte mtDNA copy number was significantly higher in gastric cancer patients than that in healthy controls (6.53 versus 4.12, P = 1.79 × 10-5), which was consistent in subgroups divided by characteristics (Table 1). In the main effect analyses, compared with those with low mtCN, the risk for gastric cancer was 1.29 (95% CI = 1.02-1.63) in the median group and 1.74 (1.39-2.18) in the high mtCN group. A significant dose-response effect was also observed (P for trend = 1.51×10-6) (Table 2). We further conducted stratified analyses based on age, sex, cigarette smoking and alcohol drinking status. We found similar associations between mtCN and gastric cancer risk in different groups (Table 3). Our previous study demonstrated a U-shaped association between peripheral blood leukocytes RTL and risk of gastric cancer [33]. We categorized participants into three RTL groups (short, median, and long) according to the RTL tertile distribution in controls. Compared with those in the median RTL group, the risk of

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gastric cancer was 1.20 (0.94-1.52) in the long RTL group and 2.11 (1.68-2.65) in the short RTL group (Supplementary Table 2). We further assessed the correlation between mtCN and RTL as well as their joint effect on gastric cancer risk. We found a negligible but significant inverse correlation between mtCN and RTL among controls (rs=-0.075, P = 0.018 for Spearman correlation test) (Figure 1). The median mtCN value in the short, median and long RTL groups was 4.91, 4.19 and 3.58, respectively. After adjusted RTL, the association between mtCN and gastric cancer risk remained statistically significant, suggesting an independent effect of these two factors on gastric cancer risk (Table 2). We then examined the joint effect of mtCN and RTL on gastric cancer risk using multivariable logistic regression models. As compared with those in the low mtCN and median RTL group, individuals in the other groups showed consistently increased risk of gastric cancer (P for trend = 1.16×10-14). Compared with those in the lowest risk group, the odds ratio for gastric cancer in the highest risk group was 4.30 (2.79-6.63) (Table 4), indicating a cumulative effect of mtCN and RTL on gastric cancer risk. However, no statistically significant multiplicative interaction between mtCN and RTL was found (P=0.133) (Table 4).

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Discussion In this study, we designed a relatively large case-control study in a Chinese population and examined the relationship between mtCN and gastric cancer risk, the correlation of mtCN and telomere length, as well as the interaction between mtCN and RTL on gastric cancer risk. Our results indicated that high mtCN might be associated with an increased risk of gastric cancer in Chinese populations, and the effects of mtCN and RTL on gastric cancer risk might be independent. Further studies are warranted to validate the potential value of these two biomarkers in risk prediction of gastric cancer. Mitochondria are eukaryotic organelles involved in many important physiological processes, including metabolism, signaling, apoptosis, cell cycle, differentiation and responsible for energy production [34]. It has been well documented that the enhanced production of mitochondrial reactive oxygen species (ROS), most notably superoxide, hydroxyl radicals, and hydrogen peroxide is a prominent by-product of cancer cell metabolism [35]. Mitochondrial aberrations, including mtDNA mutations and copy number variations, have been documented in gastric cancer [28, 36-37], suggesting that mtDNA may play a critical role in gastric tumorigenesis. Two case-control studies have reported the relationship between mtDNA copy number in leukocyte and gastric cancer risk to date. Sun et al [27] conducted a study in 132 gastric cancer cases and 125 controls of a Hispanic population and found significant association between low mtCN and increased gastric cancer risk (OR=11.00; 95% CI = 4.79-25.23), when individuals were dichotomized into high and low mtCN groups

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based on the median mtCN value in the controls. They also examined the association of leukocyte telomere length with the risk of gastric cancer, and found no significant association between short telomere length and increased gastric cancer risk. In the other study of 162 gastric cancer cases and 299 controls in a Chinese population, Liao et al [28] found no statistically significant association between leukocyte mtCN and risk of gastric cancer. However, we observed that a high mtCN was associated with a significantly increased risk of gastric cancer in Chinese population. Future prospective studies are warranted to replicate the association of leukocyte mtDNA copy number with risk of gastric cancer in Chinese population as well as in populations of different ethnicities. It was suggested that higher mtCN in cancers may be related to an increase of oxidative stress, and enhanced oxidative stress further induces mitochondrial mass and increased mtDNA copy number [38]. At the same time, ROS, generated from the increased mitochondria, can cause more oxidative damage to mitochondria and other intracellular constituents [39] and may be involved in both the initiation and promotion of carcinogenesis [40]. Telomeres are crucial in maintaining chromosomal stability and integrity through prohibiting abnormal events, such as nuclease degradation, chromosome ends fusion, and aberrant recombination [41].Telomere shortening has been implicated in the development of multiple aging-related diseases including cancer [42].Previous studies have demonstrated that mitochondria generate a large amount of reactive oxygen species as a toxic by-product of oxidative phosphorylation, which plays an

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important role in telomere shortening [43]. Reactive oxygen species generated by compromised mitochondria may oxidize proteins necessary for telomere maintenance, such as telomerase, reactive oxygen species are important mediators that link mitochondrial dysfunction and telomere shortening and loss, genomic instability, and apoptosis as well [44]. Increased mitochondrial dysfunction results in increased reactive oxygen species concentrations and subsequent activation of the DNA damage response pathway including telomere damage and erosion and the consequent enhanced p53 activation [45]. Interestingly, we found that mtCN was negatively correlated with RTL in our control population. The effects of mtCNA and RTL on gastric cancer were independent, suggesting that they could be independent risk predictors for gastric cancer. One of the highlights of this study is the relatively large sample size in our design, which allows us to discreetly evaluate the dose-response relationship between mtCN and gastric cancer risk. Second, we recruited newly diagnosed patients without any treatment before recruitment and matched cases to controls on age and gender. We measured the copy number of mtDNA in triplicate using consistent protocols by the same technicians under strict quality control. However there were some limitations of our design. Firstly, because of the case-control design, DNA was not obtained before cancer onset, we could not assess whether mtCN variations in the cases is the cause or the result of the gastric cancer. Secondly, our data did not have information on H. pylori infection status, so we could not evaluate the effects of H. pylori infection to our results. Some studies suggested that H. pylori infection may

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induce DNA damage and mutation [46], which may influence mtCN. Thirdly, we examined the mtCN and RTL in DNA from blood and the data from stomach tissues were also important to clarify the tissue-specific relationship for these two biomarkers. Fourthly, we observed differences in the distribution of drinking status but not smoking status between cases and controls, which might because of the recall bias inherent in case-control studies and the difference in populations. Finally, our results are based on one population, and further prospective studies from different populations are warranted to confirm these findings. In summary, this study indicated that subjects with high mtDNA copy number were at increased risk of gastric cancer in Chinese population. Though mtDNA copy number was negatively correlated with relative telomere length, their effects on gastric cancer risk seem independent. Our study provided clues for understanding the role of mtCN and RTL in gastric carcinogenesis. Further investigations are also needed to elucidate the functional mechanisms of mtDNA changes in the development and progression of gastric cancer. Ethics Demographic data and lifestyle related factors such as cigarette smoking and alcohol drinking were collected by face-to-face interviews, and peripheral blood samples were obtained from each study subjects. This study was granted ethical approval by the Institutional Review Board of Nanjing Medical University, and written informed consent was obtained from each study subject.

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Declarations of interests The authors state that they have no competing interests associated with this work. Author Contribution Statement G.J., Z.H., and H.S. conceived and designed the experiments; X.Z., Y.M., T.H., C.Y., and F.Y. performed the experiments and analyzed the data; Jiangbo Du, Juncheng Dai, and H.M. contributed materials/analysis tools; X.Z. and Y.M. wrote and revised the manuscript; Z.H., H.S. and G.J. revised the manuscript. All authors reviewed and approved the manuscript prior to submission.

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Funding This work was supported in part by the National Key Research Program (2016YFC1302703); National Basic Research Program (973) (2013CB910304); National Natural Science Foundation of China (81422042, 81373090); Natural Science Foundation for Distinguished Young Scholars in Jiangsu (BK20130042); Innovative Practice Training Project for Jiangsu Higher Education Institutions Undergraduate (201310312003Z); and Priority Academic Program for the Development of Jiangsu Higher Education Institutions (Public Health and Preventive Medicine).

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Reference 1.

Ferlay J, Soerjomataram I, Dikshit R, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015 Mar 1; 136(5):E359-86.

2.

Colquhoun A, Arnold M, Ferlay J, Goodman KJ, Forman D, Soerjomataram I. Global patterns of cardia and non-cardia gastric cancer incidence in 2012. Gut. 2015 Dec;64(12):1881-8.

3.

Herrera V, Parsonnet J. Helicobacter pylori and gastric adenocarcinoma. Clin Microbiol Infect. 2009 Nov;15(11):971-6.

4.

Crew KD, Neugut AI. Epidemiology of gastric cancer. World J Gastroenterol 2006 12:354–362.

5.

Fock KM, Talley N, Moayyedi P, et al. Asia-Pacific consensus guidelines on gastric cancer prevention. J Gastroenterol Hepatol 2008; 23:351–365.

6.

Chen W, Zheng R, Baade PD, et al. Cancer statistics in China, 2015. CA Cancer J Clin. 2016 Jan 25.

7.

Crew KD, Neugut AI. Epidemiology of gastric cancer. World J Gastroenterol 2006 12:354–362.

8.

Fock KM, Talley N, Moayyedi P, et al. Asia-Pacific consensus guidelines on gastric cancer prevention. J Gastroenterol Hepatol 2008; 23:351–365.

9.

Attardi G, Schatz G. Biogenesis of mitochondria. Annu Rev Cell Biol. 1988; 4:289-333.

10. Kaipparettu BA, Ma Y, Wong LJ. Functional effects of cancer mitochondria on energy metabolism and tumorigenesis: utility of transmitochondrial cybrids. Ann N Y Acad Sci. 2010 Jul;1201:137-46. 11. Attardi G, Schatz G. Biogenesis of mitochondria. Annu Rev Cell Biol. 1988; 4:289-333. 12. Veltri KL, Espiritu M, Singh G. Distinct genomic copy number in mitochondria of different mammalian organs.J Cell Physiol1990;143: 160-4 13. Xu E, Sun W, Gu J, Chow WH, Ajani JA, Wu X. Association of mitochondrial DNA copy number in peripheral blood leukocytes with risk of esophageal This article is protected by copyright. All rights reserved

 

adenocarcinoma. Carcinogenesis. 2013 Nov 34(11):2521-4. 14. Melkonian SC, Wang X, Gu J, et al. Mitochondrial DNA copy number in peripheral blood leukocytes and the risk of clear cell renal cell carcinoma. Carcinogenesis. 2015 Feb 36(2):249-55. 15. Xing J, Chen M, Wood CG, et al. Mitochondrial DNA content: its genetic heritability and association with renal cell carcinoma. J Natl Cancer Inst. 2008 Aug 6 100(15):1104-12. 16. Mondal R, Ghosh SK, Choudhury JH, et al. Mitochondrial DNA copy number and risk of oral cancer: a report from Northeast India. PLoS One. 2013 8(3):e57771. 17. Ghosh SK, Singh AS, Mondal R, Kapfo W, Khamo V, Singh YI. Dysfunction of mitochondria due to environmental carcinogens in nasopharyngeal carcinoma in the ethnic group of Northeast Indian population. Tumour Biol. 2014 Jul; 35(7):6715-24. 18. Huang B, Gao YT, Shu XO, et al. Association of leukocyte mitochondrial DNA copy number with colorectal cancer risk: Results from the Shanghai Women's Health Study. Cancer Epidemiol Biomarkers Prev. 2014 Nov 23(11):2357-65. 19. Qu F, Liu X, Zhou F, et al. Association between mitochondrial DNA content in leukocytes and colorectal cancer risk: a case-control analysis. Cancer. 2011 Jul 15 117(14):3148-55. 20. Zhou W, Zhu M, Gui M, et al. Peripheral blood mitochondrial DNA copy number is associated with prostate cancer risk and tumor burden. PLoS One. 2014 Oct 3 9(10):e109470. 21. Hofmann JN, Hosgood HD 3rd, Liu CS, et al. A nested case-control study of leukocyte mitochondrial DNA copy number and renal cell carcinoma in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. Carcinogenesis. 2014 May 35(5):1028-31. 22. Shen J, Platek M, Mahasneh A, Ambrosone CB, Zhao H. Mitochondrial copy number and risk of breast cancer: a pilot study. Mitochondrion. 2010 Jan 10(1):62-8. This article is protected by copyright. All rights reserved

 

23. Thyagarajan B, Wang R, Barcelo H, Koh WP, Yuan JM. Mitochondrial copy number is associated with colorectal cancer risk. Cancer Epidemiol Biomarkers Prev. 2012 Sep; 21(9):1574-81. 24. Bonner MR, Shen M, Liu CS, Divita M, He X, Lan Q. Mitochondrial DNA content and lung cancer risk in Xuan Wei, China. Lung Cancer. 2009 Mar; 63(3):331-4. 25. Kim C, Bassig BA, Seow WJ, et al. Mitochondrial DNA copy number and chronic lymphocytic leukemia/small lymphocytic lymphoma risk in two prospective studies. Cancer Epidemiol Biomarkers Prev. 2015 Jan 24(1):148-53. 26. Zhang J, Li D, Qu F, et al. Association of leukocyte mitochondrial DNA content with glioma risk: evidence from a Chinese case-control study. BMC Cancer. 2014 Sep 19; 14:680. 27. Sun Y, Gu J, Ajani JA, Chang DW, Wu X, Stroehlein JR. Genetic and intermediate phenotypic susceptibility markers of gastric cancer in Hispanic Americans: a case-control study. Cancer. 2014 Oct 1; 120(19):3040-8. 28. Liao LM, Baccarelli A, Shu XO, et al. Mitochondrial DNA copy number and risk of gastric cancer: a report from the Shanghai Women’s Health Study.Cancer Epidemiol Biomarkers Prev.2011 20:1944-1949. 29. Du J, Zhu X, Xie C, et al. Telomere length, genetic variants and gastric cancer risk in a Chinese population. Carcinogenesis. 2015 Sep;36(9):963-70. 30. Wong A, Cortopassi G. Reproducible quantitative PCR of mitochondrial and nuclear DNA copy number using the LightCycler. Methods Mol Biol. 2002; 197:129-37. 31. Liu CS, Cheng WL, Lee CF,et al. Alteration in the copy number of mitochondrial DNA in leukocytes of patients with mitochondrial encephalomyopathies. Acta Neurol Scand. 2006 May; 113(5):334-41. 32. Hosnijeh FS, Lan Q, Rothman N, et al. Mitochondrial DNA copy number and future risk of B-cell lymphoma in a nested case-control study in the prospective EPIC cohort. Blood. 2014 Jul 24; 124(4):530-5. 33. Cawthon RM. Telomere measurement by quantitative PCR. Nucleic Acids Res. This article is protected by copyright. All rights reserved

 

2002 May 15;30(10):e47. 34. Wallace DC. A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine.Annu Rev Genet 2005 39: 359–407. 35. Verschoor ML, Ungard R, Harbottle A, Jakupciak JP, Parr RL, Singh G. Mitochondria and cancer: past, present, and future. Biomed Res Int 2013:612369. 36. Wu CW, Yin PH, Hung WY, et al. Mitochondrial DNA mutations and mitochondrial DNA depletion in gastric cancer. Genes Chromosomes Cancer. 2005 Sep 44(1):19-28.3 37. Lee HC, Yin PH, Lin JC, et al. Mitochondrial genome instability and mtDNA depletion in human cancers. Ann N Y Acad Sci. 2005 May 1042:109-22. 38. Lee HC, Yin PH, Lu CY, Chi CW, Wei YH. Increase of mitochondria and mitochondrial DNA in response to oxidative stress in human cells. Biochem J. 2000 Jun 1; 348 Pt 2:425-32. 39. Klaunig JE, Kamendulis LM. The role of oxidative stress in carcinogenesis. Annu Rev Pharmacol Toxicol. 2004 44:239-67. 40. Du M, Prescott J, Cornelis MC, et al. Genetic predisposition to higher body mass index or type 2 diabetes and leukocyte telomere length in the Nurses' Health Study. PLoS One. 2013;8(2):e52240. 41. Blackburn EH.Telomeres and telomerase: the means to the end (Nobel lecture). Angew Chem Int Ed Engl. 2010 Oct 4;49(41):7405-21. 42. Calado RT, Young NS. Telomere diseases. N Engl J Med. 2009 Dec 10;361(24):2353-65. 43. Liu L, Trimarchi JR, Smith PJ, Keefe DL. Mitochondrial dysfunction leads to telomere attrition and genomic instability. Aging Cell. 2002 Oct;1(1):40-6. 44. Chaban Y, Boekema EJ, Dudkina NV. Structures of mitochondrial oxidative phosphorylation supercomplexes and mechanisms for their stabilisation. Biochim Biophys Acta. 2014 Apr;1837(4):418-26. 45. Liu J, Cao L, Chen J, et al. Bmi1 regulates mitochondrial function and the DNA damage response pathway. Nature. 2009 May 21;459(7245):387-92. This article is protected by copyright. All rights reserved

 

46. Kim JJ, Tao H, Carloni E, Leung WK, Graham DY, Sepulveda AR. Helicobacter pylori impairs DNA mismatch repair in gastric epithelial cells. Gastroenterology. 2002 Aug; 123(2):542-53.

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Table 1. The distributions of mitochondrial DNA copy number between gastric cases and controls overall or in subgroups. Variable

Controls median(IQR)

Cases Pa

median(IQR)

0.008 b

Age < 60 ≥ 60

3.76(9.23) 5.32(15.66)

0.093

Pa

P c value

0.650 b 6.74(16.79) 6.54(20.41)

0.668

6.04×10-6 0.039

Sex Male 4.18(13.41) 6.20(13.41) 2.24×10-3 0.834 0.215 Female 4.05(9.49) 7.71(19.40) 5.75×10-4 Smoking status d Never 4.19(10.62) 7.19(19.24) 6.13×10-4 0.758 0.704 Ever 4.05(14.66) 6.74(18.41) 3.83×10-3 Drinking status d Never 4.19(12.34) 7.21(17.56) 9.10×10-4 0.942 0.853 Ever 4.06(11.37) 6.27(20.46) 3.70×10-3 Tumor site cardia -5.82(18.58) -0.119 non-cardia -6.85(17.87) -IQR, interquartile range. a Kruskal-Wallis rank sum test. b Spearman-test. c Pearson chi-square test. d Individuals who smoked at least one cigarette a day for more than one year were defined as ever smokers, and those who drank twice or more per week for at least one year were considered as ever drinkers.

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Table 2. Associations between mitochondrial DNA copy number and gastric cancer risk. Cases Controls OR OR (n=984) (n=984) Pa Pb mtCN a (95%CI) (95%CI) b N (%) N (%) Low