Mitochondrial DNA The Journal of DNA Mapping, Sequencing, and Analysis
ISSN: 1940-1736 (Print) 1940-1744 (Online) Journal homepage: http://www.tandfonline.com/loi/imdn20
Mitochondrial D310 instability in Chinese lung cancer patients Xian-Zhong Chen, Yu Fang, Yan-Hai Shi, Jing-Hui Cui, Long-Yan Li, Yong-Chen Xu & Bin Ling To cite this article: Xian-Zhong Chen, Yu Fang, Yan-Hai Shi, Jing-Hui Cui, Long-Yan Li, YongChen Xu & Bin Ling (2015): Mitochondrial D310 instability in Chinese lung cancer patients, Mitochondrial DNA, DOI: 10.3109/19401736.2014.936426 To link to this article: http://dx.doi.org/10.3109/19401736.2014.936426
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Published online: 04 Sep 2015.
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Date: 06 November 2015, At: 01:36
http://informahealthcare.com/mdn ISSN: 1940-1736 (print), 1940-1744 (electronic) Mitochondrial DNA, Early Online: 1–4 ! 2014 Informa UK Ltd. DOI: 10.3109/19401736.2014.936426
SHORT COMMUNICATION
Mitochondrial D310 instability in Chinese lung cancer patients Xian-Zhong Chen1, Yu Fang2, Yan-Hai Shi3, Jing-Hui Cui4, Long-Yan Li5, Yong-Chen Xu5, and Bin Ling1
Downloaded by [Texas A & M International University] at 01:36 06 November 2015
1
Department of ICU, The Second People’s Hospital of Yunnan Province, Kunming, Yunnan Province, China, 2Department of Anesthesiology, the first affiliated hospital of Kunming Medical University, Kunming, Yunnan Province, China, 3Department of Clinical Laboratory, Shanxi Tumor Hospital, Taiyuan, Shanxi, China, 4Department of Medical Service, Unit 65176 of PLA, Dalian, Liaoning, China, and 5Department of Cardiology and Clinical Laboratory, 211 Hospital of PLA, Harbin, Heilongjiang, China Abstract
Keywords
To characterize the somatic mutation spectrum of mitochondrial DNA at D310 in Chinese lung cancer patients and evaluate its potential significance in Chinese lung cancer diagnosis, in this study, 237 samples, including lung tumor, adjacent normal tissue and blood samples of 79 lung cancer patients were analyzed. By comparing sequences of D310 between lung cancer tissues, adjacent normal tissue and blood samples, the somatic mutations at D310 were detected in 17.72% (14/79) of Chinese lung cancer patients; this implied that somatic mutations at D310 could be served as valuable biomarker for diagnostic of Chinese lung cancer. Further analyses indicated that deletion and heterogeneity were the predominant characters for somatic mutations detected at D310 of Chinese lung cancer patients.
Chinese, D310 mutation, lung cancer, mitochondrial DNA
Introduction Lung cancer was one of the major public health challenges in the world, accounting for around 30% of all cancer related death (Cao et al., 2011, Ferlay et al., 2010). And the mortality rate of lung cancer in China was approximately 400,000 patient deaths annually (Yang et al., 2009). However, it was still unclear for the mechanism of lung cancer carcinogenesis. Mitochondria plays important role on generating ATP, producing reactive oxygen species, and initiating and executing apoptosis. Thus, recent investigations have pinpointed the potential role of mitochondrial DNA (mtDNA) changes in carcinogenesis and mtDNA mutations in tumorigenesis have received much attention (Baysal, 2006; Chatterjee et al., 2006; Pereira et al., 2012; Salas et al., 2005; Schon et al., 2012; Yu, 2012 Yu et al., 2013). And more evidences have revealed the somatic mutations may be involved in carcinogenesis and tumor progression (Li & Hong, 2012; Yu, 2012). Human mtDNA genome includes a displacement region (D-loop) and coding region segments (Anderson et al., 1981; Andrews et al., 1999). The D-loop locates at nucleotide positions between 16,024 and 576, it is a non-coding region with essential elements on regulating mtDNA duplication and transcription (Li et al., 2012). MtDNA genome has a more than 10-fold of mutation rate than that of nuclear genes for inefficient DNA repair mechanism, lacking of protectease-free survival, later onset age (Tseng et al., 2006). The cive histones and higher level of reactive oxygen species (ROS) circumstance (Wallace et al., 1999). The high incidence of somatic mtDNA mutations were identified in
Correspondence: Bin Ling, Department of ICU, The Second People’s Hospital of Yunnan Province, Kunming, Yunnan Province, China. Tel/Fax: +86 871 6512 5707. E-mail: [email protected]
History Received 9 May 2014 Revised 10 June 2014 Accepted 15 June 2014 Published online 10 July 2014
nearly all types of cancerous tissues (Hung et al., 2010; Li & Hong, 2012; Liu et al., 2001, 2012; Navaglia et al., 2006; Van et al., 2007; Wang et al., 2007; Yu, 2012; Yu et al., 2013). Thus, many researchers claimed that somatic mtDNA mutation could be used as biomarker in early cancer diagnosis (Arasaradnam et al., 2006; Cai et al., 2011; Fliss et al., 2000; Sui et al., 2006; Tseng et al., 2006). This implied that the somatic mutations can serve as a marker for early tumor diagnostic, especially the a poly-C tract located between 303 and 315 nucleotides, known as D310, for its higher frequency in tumors (Arasaradnam et al., 2006; Cai et al., 2011; Sui et al., 2006). Patients with poor comprehensive knowledge is indispensable for us to understand tumorigenesis and develop personalized therapies (Wang et al., 2013). However, there was still lacking of genetic dataset for the somatic mutations at D310 in Chinese lung cancer patients, thus, in this study, to characterize the spectrum of mtDNA somatic mutation at D310 in Chinese lung cancer patient and evaluate its potential significance in Chinese lung cancer diagnosis, 237 samples, including lung tumor, adjacent normal tissue and blood samples of 79 lung cancer patients were analyzed.
Materials and methods Tissue specimens Samples of 79 lung cancer patients including the primary lung cancerous tissue, corresponding paracancerous normal tissue and distant normal tissue of each patient were collected who received treatment at the first affiliated hospital of Kunming medical university and The second people’s hospital of Yunnan province between 2011 and 2012. The samples were handled as described in our previous work (Fang et al., 2013). All procedures were supervised and approved by human tissue research committee of our hospital, and informed consents were obtained from all participants.
2
X.-Z. Chen et al.
DNA extraction, PCR amplification and sequence analysis The genomic DNA was extracted with the standard phenol/ chloroform method, and stored at 20 C for future usage. The forward primer, 50 -GGTCTATCACCCTATTA ACCAC-30 (nucleotides 8–29) and reverse primers, 50 -TGAGGAGGTAAGCT ACATA AACT G-30 (nucleotides 598–575) were used for amplifying fragment with D310 polymorphisms. The PCR products were purified, sequenced and analyzed as described in our previous work (Fang et al., 2013).
Downloaded by [Texas A & M International University] at 01:36 06 November 2015
Verification of mtDNA mutations The purified PCR product including the somatic mutation from the cancerous tissue and blood were transferred to the pUC18 vector. The clones were selected and sequenced directly with the forward and reverse vector primers, and the sequences were compared with the revised Cambridge reference sequence (rCRS; Andrews et al., 1999).
Results and Discussion To characterize the spectrum of mtDNA somatic mutation at D310 in Chinese lung cancer patient and evaluate its potential significance in Chinese lung cancer diagnosis, we compared the sequences covering the D310 of 237 samples from 79 unrelated lung cancer patients from Yunnan Province of China. The difference of mtDNA sequences between tumor and matched normal tissues from the same patient was defined as a somatic mutation. The fragment of D310 with the consistent cytosine repeats has the concord signal as shown in Figure 1(a); in verse, the tissue with inconsistent cytosine repeats has the overlapping signal as listed in Figure 1(b). As shown in Table S1 (supporting material online), totally 14 somatic mutations at D310 were detected among 79 Chinese lung cancer patients. To consolidate the authenticity of the somatic mutations, genome DNA of different tissue for the same individual with the somatic mutation was re-extracted, independent PCR was amplified with the fragment including the cytosine repeats at D310, and the purified PCR products were transferred into the pUC18 vector, and the clones were amplified and sequenced with the common forward and reverse primers of the pUC18 vector. As shown in Figure 2, the somatic mutations of the 14 Chinese lung cancer patients were verified, which accounted 17.72% (14/79) of the investigated patients, however, the frequency of the somatic mutations detected at D310 fragment in Chinese lung cancer patients was lower than that of breast cancer (Alhomidi et al., 2013; Xu et al., 2012), especially that of squamous cell carcinoma in situ of the head and neck (63.5%, 8/13) (Ha et al., 2002),
Mitochondrial DNA, Early Online: 1–4
previous reported lung cancers (70%, 10/14) (Fliss et al., 2000), and the different sample size was the main reason causing the different frequency of somatic mutations detected at D310. And the susceptible to oxidative damage and electrophilic attack owing to the poor DNA repair system in mitochondria can accumulate mutations at D310 (Mambo et al., 2003). Together with the associations between somatic mutations detected at D310 and poor disease-free survival, a late onset age, estrogen and progesterone-negative cancers (Tseng et al., 2006), and the higher frequency of somatic mutation rates in D310 fragment of Chinese lung cancer patients, which implied that detecting the somatic mutations at D310 fragment could be used as potential valuable molecular marker for diagnostic of Chinese lung cancer. Further, we analyzed the patterns of 14 somatic mutations at D310 fragment. Our results indicated that deletion formed the predominant form of the somatic mutations at D310 fragment, which account for 78.57% (11/14) of the patients with somatic mutations. In addition, 21.43% (3/14) of which was insertion. As revealed in health individuals, the polymorphisms at D310 sequence was consistent of cytosine repeats in the same individual (Andrews et al., 1999), it ranges from seven to nine of cytosine repeats among different individuals (Ha et al., 2002). And our results indicated that all the somatic mutations were detected by comparing the sequences of lung cancer tissue and blood (distant normal tissue), and the polymorphisms at D310 sequence from the blood have the consistent of variations of cytosine repeats for the same individual, which indicated that comparing the sequences of the primary lung cancerous tissue and blood (distant normal tissue) can identify the somatic mutations efficiently, rather than comparing the sequences of the primary lung cancerous tissue, corresponding paracancerous normal tissue and distant normal tissue of each patient. However, the somatic mutations at D310 were inconsistent of the cytosine among lung cancer tissue, most of which were at the heterogeneity status, which indicated that the heterogeneity was another character of the somatic mutations for Chinese lung cancer patients. In summary, our results indicated that the somatic mutations at D310 accounted 17.72% of all the investigated Chinese lung cancer patients, which implied that detecting the somatic mutations at D310 could be served as valuable biomarker for diagnostic of Chinese lung cancer; and detecting the differences of D310 repeats between the primary lung cancerous tissue and distant normal tissue were an efficient method for checking the somatic mutations. Deletion formed the major form of the somatic mutations and heterogeneity was another character of D310 repeats for the tumor tissue.
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12C Figure 1. Concordant (A) and inconsistent (B) sequences results between tumor and distant normal tissue.
D310 of Chinese lung cancer patients
DOI: 10.3109/19401736.2014.936426
Downloaded by [Texas A & M International University] at 01:36 06 November 2015
Figure 2. mtDNA somatic mutations detected at D310 fragment in Chinese lung cancer patients. Sequencing results of the lung cancer tissue and distant normal tissue from the same patient. And the positions were marked with the red arrows. The mutations were recorded by comparing with rCRS.
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Acknowledgements
References
We thank the volunteers for participating in the project.
Alhomidi MA, Vedicherla B, Movva S, Rao PK, Ahuja YR, Hasan Q. (2013). Mitochondrial D310 instability in Asian Indian breast cancer patients. Tumour Biol 34:2427–32. Anderson S, Bankier AT, Barrell BG, De Bruijn MH, Coulson AR, Drouin J, Eperon IC, et al. (1981). Sequence and organization of the human mitochondrial genome. Nature 290:457–65.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
Downloaded by [Texas A & M International University] at 01:36 06 November 2015
4
X.-Z. Chen et al.
Mitochondrial DNA, Early Online: 1–4
Andrews RM, Kubacka I, Chinnery PF, Lightowlers RN, Turnbull DM, Howell N. (1999). Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat Genet 23:147. Arasaradnam RP, Greaves LC, Commane D, Mathers JC, Taylor RW, Turnbull DM. (2006). Mitochondrial DNA (MTDNA) mutations in human colonic crypts: A novel biomarker of colorectal cancer. Gut 55: A24–4. Baysal BE. (2006). Role of mitochondrial mutations in cancer. Endocrine Pathology 17:203–11. Cai FF, Kohler C, Zhang B, Chen WJ, Barekati Z, Garritsen HS, Lenner P, et al. (2011). Mutations of mitochondrial DNA as potential biomarkers in breast cancer. Anticancer res 31:4267–71. Cao C, Zhang YM, Wang R, Sun SF, Chen ZB, Ma HY, Yu YM, et al. (2011). Excision repair cross complementation group 1 polymorphisms and lung cancer risk: a meta-analysis. Chin Med J (Engl) 124:2203–8. Chatterjee A, Mambo E, Sidransky D. (2006). Mitochondrial DNA mutations in human cancer. Oncogene 25:4663–74. Fang Y, Huang J, Zhang J, Wang J, Qiao F, Chen HM, Hong ZP. (2013). Detecting the somatic mutations spectrum of Chinese lung cancer by analyzing the whole mitochondrial DNA genomes. Mitochondrial DNA, doi:10.3109/19401736.2013.823168. Ferlay J, Shin HR, Bray F, Forman D, Mathers C. Parkin DM. (2010). Estimates of worldwide burden of cancer in 2008: GLOBOCAN (2008). Int J Cancer 127:2893–917. Fliss MS, Usadel H, Cabellero OL, Wu L, Buta MR, Eleff SM, Jen J. Sidransky D. (2000). Facile detection of mitochondrial DNA mutations in tumors and bodily fluids. Science 287:2017–19. Ha PK, Tong BC, Westra WH, Sanchez-Cespedes M, Parrella P, Zahurak M, Sidransky D. Califano JA. (2002). Mitochondrial C-tract alteration in premalignant lesions of the head and neck a marker for progression and clonal proliferation. Clin cancer res 8:2260–5. Hung WY, Wu CW, Yin PH, Chang CJ, Li AF, Chi CW, Wei YH, Lee HC. (2010). Somatic mutations in mitochondrial genome and their potential roles in the progression of human gastric cancer. Biochim Biophys Acta 1800:264–70. Li H. Hong ZH. (2012). Mitochondrial DNA mutations in human tumor cells. Oncol Lett 4:868–72. Li H, Liu D, Lu J. Bai Y. (2012). Physiology and pathophysiology of mitochondrial DNA. Adv Exp Med Biol 942:39–51. Liu SA, Jiang RS, Chen FJ, Wang WY, Lin JC. (2012). Somatic mutations in the D-loop of mitochondrial DNA in oral squamous cell carcinoma. Eur Arch Otorhinolaryngol 269:1665–70. Liu VW, Shi HH, Cheung AN, Chiu PM, Leung TW, Nagley P, Wong LC, Ngan HY. (2001). High incidence of somatic mitochondrial DNA mutations in human ovarian carcinomas. Cancer Res 61:5998–6001. Mambo E, Gao X, Cohen Y, Guo Z, Talalay P, Sidransky D. (2003). Electrophile and oxidant damage of mitochondrial DNA leading to
rapid evolution of homoplasmic mutations. Proc Natl Acad Sci USA 100:1838–43. Navaglia F, Basso D, Fogar P, Sperti C, Greco E, Zambon CF, Stranges A, et al. (2006). Mitochondrial DNA D-loop in pancreatic cancer: Somatic mutations are epiphenomena while the germline 16519 T variant worsens metabolism and outcome. Am J Clin Pathol 126:593–601. Pereira L, Soares P, Maximo V. Samuels DC. (2012). Somatic mitochondrial DNA mutations in cancer escape purifying selection and high pathogenicity mutations lead to the oncocytic phenotype: Pathogenicity analysis of reported somatic mtDNA mutations in tumors. BMC Cancer 12:53. Salas A, Yao YG, Macaulay V, Vega A, Carracedo A. Bandelt HJ. (2005). A critical reassessment of the role of mitochondria in tumorigenesis. PLoS Med 2:e296. Schon EA, Dimauro S. Hirano M. (2012). Human mitochondrial DNA: Roles of inherited and somatic mutations. Nat Rev Genet 13:878–90. Sui G, Zhou S, Wang J, Canto M, Lee EE, Eshleman JR, Montgomery EA, et al. (2006). Mitochondrial DNA mutations in preneoplastic lesions of the gastrointestinal tract: a biomarker for the early detection of cancer. Mol Cancer 5:73–81. Tseng LM, Yin PH, Chi CW, Hsu CY, Wu CW, Lee LM, Wei YH, Lee HC. (2006). Mitochondrial DNA mutations and mitochondrial DNA depletion in breast cancer. Genes Chromosomes Cancer 45: 629–38. Van Trappen PO, Cullup T, Troke R, Swann D, Shepherd JH, Jacobs IJ, Gayther SA, Mein CA. (2007). Somatic mitochondrial DNA mutations in primary and metastatic ovarian cancer. Gynecol Oncol 104:129–33. Wallace DC, Brown MD, Lott MT. (1999). Mitochondrial DNA variation in human evolution and disease. Gene 238:211–30. Wang CY, Wang HW, Yao YG, Kong QP, Zhang YP. (2007). Somatic mutations of mitochondrial genome in early stage breast cancer. Int J Cancer 121:1253–6. Wang Q, Jia P, Li F, Chen H, Ji H, Hucks D, Dahlman KB, et al. (2013). Detecting somatic point mutations in cancer genome sequencing data: A comparison of mutation callers. Genome Med 5:91–98. Xu C, Tran-Thanh D, Ma C, May K, Jung J, Vecchiarelli J. Done SJ. (2012). Mitochondrial D310 mutations in the early development of breast cancer. British J Cancer 106:1506–11. Yang L, Yang G, Zhou M, Smith M, Ge H, Boreham J, Hu Y, et al. (2009). Body mass index and mortality from lung cancer in smokers and nonsmokers: A nationally representative prospective study of 220,000 men in China. Int J Cancer 125:2136–43. Yu M. (2012). Somatic mitochondrial DNA mutations in human cancers. Adv Clin Chem 57:99–138. Yu M, Wan Y. Zou Q. (2013). Somatic mitochondrial DNA mutations in Chinese patients with osteosarcoma. Int J Exp Pathol doi: 10.1111/ iep.12015.
Supplementary material available online. Supplementary Table S1.
ISSN: 1940-1736 (Print) 1940-1744 (Online) Journal homepage: http://www.tandfonline.com/loi/imdn20
Mitochondrial D310 instability in Chinese lung cancer patients Xian-Zhong Chen, Yu Fang, Yan-Hai Shi, Jing-Hui Cui, Long-Yan Li, Yong-Chen Xu & Bin Ling To cite this article: Xian-Zhong Chen, Yu Fang, Yan-Hai Shi, Jing-Hui Cui, Long-Yan Li, YongChen Xu & Bin Ling (2015): Mitochondrial D310 instability in Chinese lung cancer patients, Mitochondrial DNA, DOI: 10.3109/19401736.2014.936426 To link to this article: http://dx.doi.org/10.3109/19401736.2014.936426
View supplementary material
Published online: 04 Sep 2015.
Submit your article to this journal
Article views: 19
View related articles
View Crossmark data
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=imdn20 Download by: [Texas A & M International University]
Date: 06 November 2015, At: 01:36
http://informahealthcare.com/mdn ISSN: 1940-1736 (print), 1940-1744 (electronic) Mitochondrial DNA, Early Online: 1–4 ! 2014 Informa UK Ltd. DOI: 10.3109/19401736.2014.936426
SHORT COMMUNICATION
Mitochondrial D310 instability in Chinese lung cancer patients Xian-Zhong Chen1, Yu Fang2, Yan-Hai Shi3, Jing-Hui Cui4, Long-Yan Li5, Yong-Chen Xu5, and Bin Ling1
Downloaded by [Texas A & M International University] at 01:36 06 November 2015
1
Department of ICU, The Second People’s Hospital of Yunnan Province, Kunming, Yunnan Province, China, 2Department of Anesthesiology, the first affiliated hospital of Kunming Medical University, Kunming, Yunnan Province, China, 3Department of Clinical Laboratory, Shanxi Tumor Hospital, Taiyuan, Shanxi, China, 4Department of Medical Service, Unit 65176 of PLA, Dalian, Liaoning, China, and 5Department of Cardiology and Clinical Laboratory, 211 Hospital of PLA, Harbin, Heilongjiang, China Abstract
Keywords
To characterize the somatic mutation spectrum of mitochondrial DNA at D310 in Chinese lung cancer patients and evaluate its potential significance in Chinese lung cancer diagnosis, in this study, 237 samples, including lung tumor, adjacent normal tissue and blood samples of 79 lung cancer patients were analyzed. By comparing sequences of D310 between lung cancer tissues, adjacent normal tissue and blood samples, the somatic mutations at D310 were detected in 17.72% (14/79) of Chinese lung cancer patients; this implied that somatic mutations at D310 could be served as valuable biomarker for diagnostic of Chinese lung cancer. Further analyses indicated that deletion and heterogeneity were the predominant characters for somatic mutations detected at D310 of Chinese lung cancer patients.
Chinese, D310 mutation, lung cancer, mitochondrial DNA
Introduction Lung cancer was one of the major public health challenges in the world, accounting for around 30% of all cancer related death (Cao et al., 2011, Ferlay et al., 2010). And the mortality rate of lung cancer in China was approximately 400,000 patient deaths annually (Yang et al., 2009). However, it was still unclear for the mechanism of lung cancer carcinogenesis. Mitochondria plays important role on generating ATP, producing reactive oxygen species, and initiating and executing apoptosis. Thus, recent investigations have pinpointed the potential role of mitochondrial DNA (mtDNA) changes in carcinogenesis and mtDNA mutations in tumorigenesis have received much attention (Baysal, 2006; Chatterjee et al., 2006; Pereira et al., 2012; Salas et al., 2005; Schon et al., 2012; Yu, 2012 Yu et al., 2013). And more evidences have revealed the somatic mutations may be involved in carcinogenesis and tumor progression (Li & Hong, 2012; Yu, 2012). Human mtDNA genome includes a displacement region (D-loop) and coding region segments (Anderson et al., 1981; Andrews et al., 1999). The D-loop locates at nucleotide positions between 16,024 and 576, it is a non-coding region with essential elements on regulating mtDNA duplication and transcription (Li et al., 2012). MtDNA genome has a more than 10-fold of mutation rate than that of nuclear genes for inefficient DNA repair mechanism, lacking of protectease-free survival, later onset age (Tseng et al., 2006). The cive histones and higher level of reactive oxygen species (ROS) circumstance (Wallace et al., 1999). The high incidence of somatic mtDNA mutations were identified in
Correspondence: Bin Ling, Department of ICU, The Second People’s Hospital of Yunnan Province, Kunming, Yunnan Province, China. Tel/Fax: +86 871 6512 5707. E-mail: [email protected]
History Received 9 May 2014 Revised 10 June 2014 Accepted 15 June 2014 Published online 10 July 2014
nearly all types of cancerous tissues (Hung et al., 2010; Li & Hong, 2012; Liu et al., 2001, 2012; Navaglia et al., 2006; Van et al., 2007; Wang et al., 2007; Yu, 2012; Yu et al., 2013). Thus, many researchers claimed that somatic mtDNA mutation could be used as biomarker in early cancer diagnosis (Arasaradnam et al., 2006; Cai et al., 2011; Fliss et al., 2000; Sui et al., 2006; Tseng et al., 2006). This implied that the somatic mutations can serve as a marker for early tumor diagnostic, especially the a poly-C tract located between 303 and 315 nucleotides, known as D310, for its higher frequency in tumors (Arasaradnam et al., 2006; Cai et al., 2011; Sui et al., 2006). Patients with poor comprehensive knowledge is indispensable for us to understand tumorigenesis and develop personalized therapies (Wang et al., 2013). However, there was still lacking of genetic dataset for the somatic mutations at D310 in Chinese lung cancer patients, thus, in this study, to characterize the spectrum of mtDNA somatic mutation at D310 in Chinese lung cancer patient and evaluate its potential significance in Chinese lung cancer diagnosis, 237 samples, including lung tumor, adjacent normal tissue and blood samples of 79 lung cancer patients were analyzed.
Materials and methods Tissue specimens Samples of 79 lung cancer patients including the primary lung cancerous tissue, corresponding paracancerous normal tissue and distant normal tissue of each patient were collected who received treatment at the first affiliated hospital of Kunming medical university and The second people’s hospital of Yunnan province between 2011 and 2012. The samples were handled as described in our previous work (Fang et al., 2013). All procedures were supervised and approved by human tissue research committee of our hospital, and informed consents were obtained from all participants.
2
X.-Z. Chen et al.
DNA extraction, PCR amplification and sequence analysis The genomic DNA was extracted with the standard phenol/ chloroform method, and stored at 20 C for future usage. The forward primer, 50 -GGTCTATCACCCTATTA ACCAC-30 (nucleotides 8–29) and reverse primers, 50 -TGAGGAGGTAAGCT ACATA AACT G-30 (nucleotides 598–575) were used for amplifying fragment with D310 polymorphisms. The PCR products were purified, sequenced and analyzed as described in our previous work (Fang et al., 2013).
Downloaded by [Texas A & M International University] at 01:36 06 November 2015
Verification of mtDNA mutations The purified PCR product including the somatic mutation from the cancerous tissue and blood were transferred to the pUC18 vector. The clones were selected and sequenced directly with the forward and reverse vector primers, and the sequences were compared with the revised Cambridge reference sequence (rCRS; Andrews et al., 1999).
Results and Discussion To characterize the spectrum of mtDNA somatic mutation at D310 in Chinese lung cancer patient and evaluate its potential significance in Chinese lung cancer diagnosis, we compared the sequences covering the D310 of 237 samples from 79 unrelated lung cancer patients from Yunnan Province of China. The difference of mtDNA sequences between tumor and matched normal tissues from the same patient was defined as a somatic mutation. The fragment of D310 with the consistent cytosine repeats has the concord signal as shown in Figure 1(a); in verse, the tissue with inconsistent cytosine repeats has the overlapping signal as listed in Figure 1(b). As shown in Table S1 (supporting material online), totally 14 somatic mutations at D310 were detected among 79 Chinese lung cancer patients. To consolidate the authenticity of the somatic mutations, genome DNA of different tissue for the same individual with the somatic mutation was re-extracted, independent PCR was amplified with the fragment including the cytosine repeats at D310, and the purified PCR products were transferred into the pUC18 vector, and the clones were amplified and sequenced with the common forward and reverse primers of the pUC18 vector. As shown in Figure 2, the somatic mutations of the 14 Chinese lung cancer patients were verified, which accounted 17.72% (14/79) of the investigated patients, however, the frequency of the somatic mutations detected at D310 fragment in Chinese lung cancer patients was lower than that of breast cancer (Alhomidi et al., 2013; Xu et al., 2012), especially that of squamous cell carcinoma in situ of the head and neck (63.5%, 8/13) (Ha et al., 2002),
Mitochondrial DNA, Early Online: 1–4
previous reported lung cancers (70%, 10/14) (Fliss et al., 2000), and the different sample size was the main reason causing the different frequency of somatic mutations detected at D310. And the susceptible to oxidative damage and electrophilic attack owing to the poor DNA repair system in mitochondria can accumulate mutations at D310 (Mambo et al., 2003). Together with the associations between somatic mutations detected at D310 and poor disease-free survival, a late onset age, estrogen and progesterone-negative cancers (Tseng et al., 2006), and the higher frequency of somatic mutation rates in D310 fragment of Chinese lung cancer patients, which implied that detecting the somatic mutations at D310 fragment could be used as potential valuable molecular marker for diagnostic of Chinese lung cancer. Further, we analyzed the patterns of 14 somatic mutations at D310 fragment. Our results indicated that deletion formed the predominant form of the somatic mutations at D310 fragment, which account for 78.57% (11/14) of the patients with somatic mutations. In addition, 21.43% (3/14) of which was insertion. As revealed in health individuals, the polymorphisms at D310 sequence was consistent of cytosine repeats in the same individual (Andrews et al., 1999), it ranges from seven to nine of cytosine repeats among different individuals (Ha et al., 2002). And our results indicated that all the somatic mutations were detected by comparing the sequences of lung cancer tissue and blood (distant normal tissue), and the polymorphisms at D310 sequence from the blood have the consistent of variations of cytosine repeats for the same individual, which indicated that comparing the sequences of the primary lung cancerous tissue and blood (distant normal tissue) can identify the somatic mutations efficiently, rather than comparing the sequences of the primary lung cancerous tissue, corresponding paracancerous normal tissue and distant normal tissue of each patient. However, the somatic mutations at D310 were inconsistent of the cytosine among lung cancer tissue, most of which were at the heterogeneity status, which indicated that the heterogeneity was another character of the somatic mutations for Chinese lung cancer patients. In summary, our results indicated that the somatic mutations at D310 accounted 17.72% of all the investigated Chinese lung cancer patients, which implied that detecting the somatic mutations at D310 could be served as valuable biomarker for diagnostic of Chinese lung cancer; and detecting the differences of D310 repeats between the primary lung cancerous tissue and distant normal tissue were an efficient method for checking the somatic mutations. Deletion formed the major form of the somatic mutations and heterogeneity was another character of D310 repeats for the tumor tissue.
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(B)
rCRS
rCRS
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12C Figure 1. Concordant (A) and inconsistent (B) sequences results between tumor and distant normal tissue.
D310 of Chinese lung cancer patients
DOI: 10.3109/19401736.2014.936426
Downloaded by [Texas A & M International University] at 01:36 06 November 2015
Figure 2. mtDNA somatic mutations detected at D310 fragment in Chinese lung cancer patients. Sequencing results of the lung cancer tissue and distant normal tissue from the same patient. And the positions were marked with the red arrows. The mutations were recorded by comparing with rCRS.
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Acknowledgements
References
We thank the volunteers for participating in the project.
Alhomidi MA, Vedicherla B, Movva S, Rao PK, Ahuja YR, Hasan Q. (2013). Mitochondrial D310 instability in Asian Indian breast cancer patients. Tumour Biol 34:2427–32. Anderson S, Bankier AT, Barrell BG, De Bruijn MH, Coulson AR, Drouin J, Eperon IC, et al. (1981). Sequence and organization of the human mitochondrial genome. Nature 290:457–65.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
Downloaded by [Texas A & M International University] at 01:36 06 November 2015
4
X.-Z. Chen et al.
Mitochondrial DNA, Early Online: 1–4
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Supplementary material available online. Supplementary Table S1.
