基于LC-MS技术的患乳腺癌猫血清代谢组学分析

doi:10.11843/j.issn.0366-6964.2020.09.027

摘要/Abstract

摘要： 本试验旨在研究患乳腺癌猫与健康猫体内差异代谢物的变化情况，并探讨差异代谢物与猫乳腺癌发生之间的关系。本试验选取临床收集的经组织病理学确诊的猫乳腺癌血清样本6例作为试验组（T组），同时选取年龄相似，品种相同的健康猫血清6例作为对照组（C组）。运用超高效液相色谱质谱联用技术（liquid chromatography-mass spectrometry，LC-MS）对两组猫的血清样本进行检测，采用无监督的主成分分析（principal component analysis，PCA）、正交偏最小二乘法-判别分析（orthogonal projections to latent structures-discriminant analysis，OPLS-DA）及学生t检验（Student's t-test）筛选出差异代谢物，然后再对筛选出的差异代谢物进行层次聚类分析（hierarchical clustering analysis，HCA），并进行差异代谢物的KEGG注释及差异代谢物的代谢通路分析。结果表明，在两组样本中共定性到159种差异代谢物。与C组相比，在T组中有49种差异代谢物出现下调，110种差异代谢物出现上调。最终共筛选出5种与猫乳腺癌发生发展密切相关的差异代谢产物。与C组相比，T组中麦角硫因（ergothioneine，EGT）和肌酸（creatine）出现下调，吲哚乙酸（indolelactic acid，IAA）、胆碱（choline）和尿酸（uric acid）出现上调。这些差异代谢物表明，在猫乳腺癌的发生过程中，机体变化涉及了甘氨酸、丝氨酸和苏氨酸代谢、精氨酸和脯氨酸代谢、组氨酸代谢、色氨酸代谢、嘌呤代谢异常和甘油磷脂代谢等多个代谢途径，为今后深入研究猫乳腺癌的发病机制开辟了一个新的思路。

关键词: 猫, 乳腺癌, LC-MS, 差异代谢产物

Abstract: The purpose of this study was to investigate the variation of differential metabolites in mammary carcinomas bearing cats and healthy cats, and to explore the relationship between differential metabolites and the occurrence of feline mammary carcinomas. In this study, 6 feline mammary carcinomas serum samples were selected as test (T) group. Meanwhile, 6 healthy cats with similar age and same breed were selected as control (C) group. Serum samples were detected by ultra-high performance liquid tandem chromatography quadrupole time of flight mass spectrometry (LC-MS). Differential metabolites were screened by principal component analysis (PCA), orthogonal projections to latent structures-discriminant analysis (OPLS-DA) and Student's t-test. Then the hierarchical clustering analysis (HCA) was carried out for the screened differential metabolites. The KEGG annotation and the metabolic pathway of differential metabolites were analyzed. The results showed that a total of 159 differential metabolites were identified in the 2 groups. Compared with C group, 49 differential metabolites were down-regulated and 110 differential metabolites were up-regulated in T group. Finally, a total of 5 differential metabolites which closely related to feline mammary carcinomas were selected. Ergothioneine (EGT) and creatine in T group were down-regulated, while indolelactic acid (IAA), choline and uric acid were up-regulated compared to C group. These differential metabolites indicated that during the development of feline mammary carcinomas, the body changes involved multiple metabolic pathways, such as glycine, serine and threonine metabolism, arginine and proline metabolism, histidine metabolism, tryptophan metabolism, purine metabolism and glycerophospholipid metabolism. This study provides a new idea for further research of the pathogenesis of feline mammary carcinomas.

Key words: feline, mammary carcinomas, LC-MS, differential metabolites

中图分类号:

S858.293.7.26

韦人月, 王峥, 周志新, 邓朝阳, 侯凯文, 郑家三. 基于LC-MS技术的患乳腺癌猫血清代谢组学分析[J]. 畜牧兽医学报, 2020, 51(9): 2293-2301.

WEI Renyue, WANG Zheng, ZHOU Zhixin, DENG Chaoyang, HOU Kaiwen, ZHENG Jiasan. Serum Metabolomics Analysis of Feline Mammary Carcinomas Based on LC-MS Techniques[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(9): 2293-2301.

参考文献 36

[1]	CUNHA S,CORGOZINHO K,JUSTEN H,et al.Survival and disease-free interval of cats with mammary carcinoma treated with chain mastectomy[J].Acta Sci Vet,2016,44(1):1349.
[2]	HAHN K A,BRAVO L,AVENELL J S.Feline breast carcinoma as a pathologic and therapeutic model for human breast cancer[J].In Vivo,1994,8(5):825-828.
[3]	OVERLEY B,SHOFER F S,GOLDSCHMIDT M H,et al.Association between ovarihysterectomy and feline mammary carcinoma[J].J Vet Intern Med,2005,19(4):560-563.
[4]	HAYES H M JR,MILNE K L,MANDELL C P.Epidemiological features of feline mammary carcinoma[J].Vet Rec,1981,108(22):476-479.
[5]	WEIJER K,HART A A.Prognostic factors in feline mammary carcinoma[J].J Natl Cancer Inst,1983,70(4):709-716.
[6]	TOGNI M,MASUDA E K,KOMMERS G D,et al.A retrospective study of 207 cases of mammary tumors in queens[J].Pesq Vet Bras,2013,33(3):353-358.
[7]	何柳,吴文玉,赵德明,等.猫乳腺单纯癌病理诊断报告病例[J].中国兽医杂志,2013,49(6):62-63.HE L,WU W Y,ZHAO D M,et al.A case report of pathological diagnosis of cat breast simple carcinoma[J].Chinese Journal of Veterinary Medicine,2013,49(6):62-63.(in Chinese)
[8]	ZAPPULLI V,RASOTTO R,CALIARI D,et al.Prognostic evaluation of feline mammary carcinomas:a review of the literature[J].Vet Pathol,2015,52(1):46-60.
[9]	SOARES M,MADEIRA S,CORREIA J,et al.Molecular based subtyping of feline mammary carcinomas and clinicopathological characterization[J].Breast,2016,27:44-51.
[10]	CUI P F,HUANG C H,GUO J Q,et al.Metabolic profiling of tumors,sera,and skeletal muscles from an orthotopic murine model of gastric cancer associated-cachexia[J].J Proteome Res,2019,18(4):1880-1892.
[11]	LI W J,HONG B,LI Q,et al.An integrated serum and urinary metabonomic research of Rhizoma Curcumae-Rhizoma Sparganii drug pair in hysteromyoma rats based on UPLC-Q-TOF-MS analysis[J].J Ethnopharmacol,2019,231:374-385.
[12]	MANISCALCO L,GUIL-LUNA S,IUSSICH S,et al.Expression of the short form of RON/STK in feline mammary carcinoma[J].Vet Pathol,2019,56(2):220-229.
[13]	CHANG Y C,CHUANG H L,YIN J H,et al.Signi-ficance of sphingosine kinase 1 expression in feline mammary tumors[J].BMC Vet Res,2019,15:155.
[14]	DAGHER E,ABADIE J,LOUSSOUARN D,et al.Feline invasive mammary carcinomas:prognostic value of histological grading[J].Vet Pathol,2019,56(5):660-670.
[15]	GUNDIM L F,WILSON T M,SOARES N P,et al.Her-2 expression in feline mammary lesions[J].Acta Vet Brno,2019,88(1):43-47.
[16]	FERREIRA D,MARTINS B,SOARES M,et al.Gene expression association study in feline mammary carcinomas[J].PLoS One,2019,14(8):e0221776.
[17]	CHEN E M,LU J,CHEN D Y,et al.Dynamic changes of plasma metabolites in pigs with GalN-induced acute liver failure using GC-MS and UPLC-MS[J].Biomed Pharmacother,2017,93:480-489.
[18]	DE SOUSA E B,DOS SANTOS JUNIOR G C,DUARTE M E L,et al.Metabolomics as a promising tool for early osteoarthritis diagnosis[J].Braz J Med Biol Res,2017,50(11):e6485.
[19]	YOSHIDA S,SHIME H,FUNAMI K,et al.The anti-oxidant ergothioneine augments the immunomodulatory function of TLR agonists by direct action on macrophages[J].PLoS One,2017,12(1):e0169360.
[20]	CHEAH I K,HALLIWELL B.Ergothioneine;antioxidant potential,physiological function and role in disease[J].Biochim Biophys Acta,2012,1822(5):784-793.
[21]	PERSKY A M,BRAZEAU G A,HOCHHAUS G.Pharmacokinetics of the dietary supplement creatine[J].Clin Pharmacokinet,2003,42(6):557-574.
[22]	JOVÉ M,COLLADO R,QUILES J L,et al.A plasma metabolomic signature discloses human breast cancer[J].Oncotarget,2017,8(12):19522-19533.
[23]	COOPER R,NACLERIO F,ALLGROVE J,et al.Creatine supplementation with specific view to exercise/sports performance:an update[J].J Int Soc Sports Nutr,2012,9(1):33-43.
[24]	DEMINICE R,ROSA F T,FRANCO G S,et al.Effects of creatine supplementation on oxidative stress and inflammatory markers after repeated-sprint exercise in humans[J].Nutrition,2013,29(9):1127-1132.
[25]	CAMPOS-FERRAZ P L,GUALANO B,DAS NEVES W,et al.Exploratory studies of the potential anti-cancer effects of creatine[J].Amino Acids,2016,48(8):1993-2001.
[26]	高宁.吲哚乙酸/辣根过氧化物酶对人涎腺腺样囊性癌发生发展作用的实验研究[D].郑州:郑州大学,2017.GAO N.Experimental Study on the impact of indole acetic acid/horseradish peroxidase on the occurrence and development of salivary adenoid cystic carcinoma (SACC)[D].Zhengzhou:Zhengzhou University,2017.(in Chinese)
[27]	李万君.尿中吲哚乙酸测定对诊断早期胃癌的探索[J].白求恩医科大学学报,1979(3):90-92.LI W J.Detection of indoleacetic acid in urine for the diagnosis of early gastric cancer[J].Journal of Norman Bethune University of Medical Science,1979(3):90-92.(in Chinese)
[28]	宁波市第二医院肿瘤科肿瘤实验室.测定尿中吲哚乙酸对诊断胃癌的初步探讨[J].肿瘤防治研究,1975,3(2):27-33.Department of Oncology,Ningbo Second Hospital. Preliminary study on determination of indoleacetic acid in urine for diagnosis of gastric cancer[J].Cancer Research on Prevention and Treatment,1975,3(2):27-33.(in Chinese)
[29]	ZONI E,MINOLI M,BOVET C,et al.Preoperative plasma fatty acid metabolites inform risk of prostate cancer progression and may be used for personalized patient stratification[J].BMC Cancer,2019,19(1):1216-1233.
[30]	KATZ-BRULL R,MARGALIT R,DEGANI H.Differential routing of choline in implanted breast cancer and normal organs[J].Magn Reson Med,2001,46(1):31-38.
[31]	KATZ-BRULL R,SEGER D,RIVENSON-SEGAL D,et al.Metabolic markers of breast cancer:enhanced choline metabolism and reduced choline-ether-phospholipid synthesis[J].Cancer Res,2002,62(7):1966-1970.
[32]	ACKERSTAFF E,GLUNDE K,BHUJWALLA Z M.Choline phospholipid metabolism:a target in cancer cells?[J].J Cell Biochem,2003,90(3):525-533.
[33]	周建辉,陈香美.尿酸在临床疾病中的意义[J].实用医学杂志,2004,20(3):337-338.ZHOU J H,CHEN X M.The significance of uric acid in clinical diseases[J].The Journal of Practical Medicine,2004,20(3):337-338.(in Chinese)
[34]	SPITSIN S,KOPROWSKI H.Role of uric acid in multiple sclerosis[J].Curr Top Microbiol Immunol,2008,318:325-342.
[35]	MARTINON F,PÉTRILLI V,MAYOR A,et al.Gout-associated uric acid crystals activate the NALP3 inflammasome[J].Nature,2006,440(7081):237-241.
[36]	WANG Y H,MA X L,SU C,et al.Uric acid enhances the antitumor immunity of dendritic cell-based vaccine[J].Sci Rep,2015,5:16427-16437.