肥胖小鼠胰腺组织肿瘤相关基因突变频率的分析

doi:10.3969/j.issn.1004-616x.2018.03.004

摘要/Abstract

摘要： 目的：检测肥胖小鼠胰腺组织中癌基因Kras，抑癌基因Trp53、Smad4的突变，初步探讨肥胖与胰腺组织肿瘤相关基因突变的关系。方法：将雄性C57BL/6小鼠分为两组，分别以普通食物和高脂食物饲养24周，测量其体质量、进食量、体成分和血脂水平。提取高脂食物组小鼠胰腺组织的DNA，设计引物并通过高保真DNA聚合酶扩增Kras基因第2外显子，Trp53基因第8外显子，Smad4基因第9外显子的基因片段，进行DNA测序。通过实时荧光定量PCR（qPCR）和免疫组化方法检测细胞增殖标志基因Ki67在胰腺内的表达水平。结果：24周高脂食物饲养引起小鼠肥胖。与普通食物组相比，高脂食物组小鼠的体质量、摄入单位质量饲料的体质量增长量、体脂质量、肝体比、附睾脂肪脂体比均显著增高（P均 < 0.05），血浆中甘油三酯、总胆固醇和低密度脂蛋白胆固醇的水平也显著上升（P < 0.05）。qPCR结果显示，高脂食物组小鼠胰腺组织Ki67 mRNA的水平显著升高（P < 0.05）。免疫组化结果显示，高脂食物组小鼠胰腺内存在多个Ki67阳性的胰腺导管细胞，提示胰腺细胞增殖活性升高。同时，检测到胰腺癌促癌基因Kras在肥胖小鼠中Kras基因CDS序列的第96位碱基由T突变为C（突变频率为2%）。结论：肥胖可能会增加胰腺组织中Kras基因的不稳定性，从而起到促进胰腺癌发生、发展的作用。

关键词: 肥胖, 胰腺癌, 基因突变, 小鼠

Abstract: OBJECTIVE: To detect expression of mutations in tumor-related genes,such as Kras,Trp53 and Smad4 in pancreatic tissues of diet-induced obese mice and to explore whether obesity is associated with genome instability in the pancreas. METHODS: Male C57BL/6 mice were fed normal chow diet and high-fat diet for 24 weeks. The body weight,food intake,body composition and plasma lipid were measured. Pancreatic genomic DNA was extracted and the exon 2 of Kras,exon 8 of Trp53 and exon 9 of Smad4 were amplified by using high-fidelity PCR. The PCR products were then subjected to the Sanger sequencing protocol. Cell proliferation was assessed by qPCR and immunohistochemistry for Ki67. RESULTS: Intake of 24 weeks of high-fat diet led to severe obesity in mice. High-fat diet feeding increased the body weight,fat mass,lean mass,hepatosomatic index and body-fat ratio of epididymal (P < 0.05). Body weight gain in high-fat diet group was significantly higher than those of chow diet group from eating the same weight of food(P < 0.05). Plasma lipid such as triglyceride,total cholesterol and low density lipoprotein cholesterol were significantly higher in high-fat diet group(P < 0.05). Moreover,the level of Ki67 mRNA was increased in the high-fat diet group(P < 0.05). There were several Ki67 positive cells in pancreatic duct,indicating that proliferation of pancreatic cells in obese mice were elevated. We detected 1 locus mutation in the Kras gene in 1 out of 50 samples (rate:2%). There was no mutation in the other two genes. CONCLUSION: Obesity resulted in instability of Kras gene in pancreatic tissue,which could play a role in the initiation and progression of pancreatic cancer.

Key words: obesity, pancreatic cancer, mutation, mice

中图分类号:

R730.2

王萍, 刘冰峰, 杨云风, 蔡羽薇, 谭娜, 姚丽娟, 罗燃燃, 张果. 肥胖小鼠胰腺组织肿瘤相关基因突变频率的分析[J]. 癌变·畸变·突变, 2018, 30(3): 182-187.

WANG Ping, LIU Bingfeng, YANG Yunfeng, CAI Yuwei, TAN Na, YAO Lijuan, LUO Ranran, ZHANG Guo. Mutation frequencies of tumor-related genes in the pancreatic tissues of diet-induced obese mice[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2018, 30(3): 182-187.

参考文献

[1] YANG G,WANG Y,ZENG Y,et al. Rapid health transition in China,1990-2010:findings from the Global Burden of Disease Study 2010[J]. Lancet,2013,381(9882):1987-2015.
[2] CHEN W,ZHENG R,BAADE P D,et al. Cancer statistics in china,2015[J]. CA Cancer J Clin,2016,66(2):115-132.
[3] COLLABORATION N C. Trends in adult body-mass index in 200 countries from 1975 to 2014:a pooled analysis of 1698 population-based measurement studies with 19.2 million participants[J]. Lancet,2016,387(10026):1377-1396.
[4] AUNE D,GREENWOOD D C,CHAN D S,et al. Body mass index,abdominal fatness and pancreatic cancer risk:a systematic review and non-linear dose-response meta-analysis of prospective studies[J]. Ann Oncol,2012,23(4):843-852.
[5] LI D,MORRIS J S,LIU J,et al. Body mass index and risk,age of onset,and survival in patients with pancreatic cancer[J]. JAMA,2009,301(24):2553-2562.
[6] FONT-BURGADA J,SUN B,KARIN M. Obesity and cancer:the oil that feeds the flame[J]. Cell Metab,2016,23(1):48-62.
[7] IACOBUZIO-DONAHUE C A,VELCULESCU V E,WOLFGANG C L,et al. Genetic basis of pancreas cancer development and progression:insights from whole-exome and whole-genome sequencing[J]. Clin Cancer Res,2012,18(16):4257-4265.
[8] IACOBUZIO-DONAHUE C A. Genetic evolution of pancreatic cancer:lessons learnt from the pancreatic cancer genome sequencing project[J]. Gut,2012,61(7):1085-1094.
[9] PEREZ-MANCERA P A,GUERRA C,BARBACID M,et al. What we have learned about pancreatic cancer from mouse models[J]. Gastroenterology,2012,142(5):1079-1092.
[10] FORBES S A,BEARE D,BOUTSELAKIS H. Catalogue of somatic mutations in cancer:Cosmic v83[DB/OL]. 2017,http://cancer.sanger.ac.uk/cosmic.
[11] KYRGIOU M,KALLIALA I,MARKOZANNES G,et al. Adiposity and cancer at major anatomical sites:umbrella review of the literature[J]. BMJ,2017,356:j477.
[12] INCIO J,TAM J,RAHBARI N N,et al. PlGF/VEGFR-1 signaling promotes macrophage polarization and accelerated tumor progression in obesity[J]. Clin Cancer Res,2016,22(12):2993-3004.
[13] INCIO J,LIU H,SUBOJ P,et al. Obesity-induced inflammation and desmoplasia promote pancreatic cancer progression and resistance to chemotherapy[J]. Cancer Discov,2016,6(8):852-869.
[14] GALLAGHER E J,LEROITH D. The proliferating role of insulin and insulin-like growth factors in cancer[J]. Trends Endocrinol Metab,2010,21(10):610-618.
[15] SPENTZOS D,CANNISTRA S A,GRALL F,et al. IGF axis gene expression patterns are prognostic of survival in epithelial ovarian cancer[J]. Endocr Relat Cancer,2007,14(3):781-790.
[16] HAILEY J,MAXWELL E,KOUKOURAS K,et al. Neutralizing anti-insulin-like growth factor receptor 1 antibodies inhibit receptor function and induce receptor degradation in tumor cells[J]. Mol Cancer Ther,2002,1(14):1349-1353.
[17] SACHDEV D,LI S L,HARTELL J S,et al. A chimeric humanized single-chain antibody against the type I insulin-like growth factor (IGF) receptor renders breast cancer cells refractory to the mitogenic effects of IGF-I[J]. Cancer Res,2003,63(3):627-635.
[18] GOETSCH L,GONZALEZ A,LEGER O,et al. A recombinant humanized anti-insulin-like growth factor receptor type I antibody (h7C10) enhances the antitumor activity of vinorelbine and anti-epidermal growth factor receptor therapy against human cancer xenografts[J]. Int J Cancer,2005,113(2):316-328.
[19] RAY A,NKHATA K J,CLEARY M P. Effects of leptin on human breast cancer cell lines in relationship to estrogen receptor and HER2 status[J]. Int J Oncol,2007,30(6):1499-1509.
[20] LIU H,MA Q,LI J. High glucose promotes cell proliferation and enhances GDNF and RET expression in pancreatic cancer cells[J]. Mol Cell Biochem,2011,347(1/2):95-101.
[21] WANG Y,LAM J B,LAM K S,et al. Adiponectin modulates the glycogen synthase kinase-3beta/beta-catenin signaling pathway and attenuates mammary tumorigenesis of MDA-MB-231 cells in nude mice[J]. Cancer Res,2006,66(23):11462-11470.
[22] TANG C H,LU M E. Adiponectin increases motility of human prostate cancer cells via adipoR,p38,AMPK,and NF-kappaB pathways[J]. Prostate,2009,69(16):1781-1789.
[23] JACKSON L,WAHLI W,MICHALIK L,et al. Potential role for peroxisome proliferator activated receptor (PPAR) in preventing colon cancer[J]. Gut,2003,52(9):1317-1322.
[24] DONMEZ-ALTUNTAS H,SAHIN F,BAYRAM F,et al. Evaluation of chromosomal damage,cytostasis,cytotoxicity,oxidative DNA damage and their association with body-mass index in obese subjects[J]. Mutat Res Genet Toxicol Environ Mutagen,2014,771:30-36.
[25] FERNANDEZ-SANCHEZ A,MADRIGAL-SANTILLAN E,BAUTISTA M,et al. Inflammation,oxidative stress,and obesity[J]. Int J Mol Sci,2011,12(5):3117-3132.
[26] HEYDARI A R,UNNIKRISHNAN A,LUCENTE L V,et al. Caloric restriction and genomic stability[J]. Nucleic Acids Res,2007,35(22):7485-7496.
[27] SPINDLER S R. Rapid and reversible induction of the longevity,anticancer and genomic effects of caloric restriction[J]. Mech Ageing Dev,2005,126(9):960-966.
[28] BIANKIN A V,WADDELL N,KASSAHN K S,et al. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes[J]. Nature,2012,491(7424):399-405.
[29] KANDA M,MATTHAEI H,WU J,et al. Presence of somatic mutations in most early-stage pancreatic intraepithelial neoplasia[J]. Gastroenterology,2012,142(4):730-733.
[30] JONES S,ZHANG X,PARSONS D W,et al. Core signaling pathways in human pancreatic cancers revealed by global genomic analyses[J]. Science,2008,321(5897):1801-1806.
[31] HOSODA W,CHIANCHIANO P,GRIFFIN J F,et al. Genetic analyses of isolated high-grade pancreatic intraepithelial neoplasia (HG-PanIN) reveal paucity of alterations in TP53 and SMAD4[J]. J Pathol,2017,242(1):16-23.