联合iTRAQ-LC MS;MS技术构建下咽鳞癌转录调控网络（可编辑）

联合iTRAQ-LC MS;MS技术构建下咽鳞癌转录调控网络（可编辑） 联合iTRAQ-LC MS;MS技术构建下咽鳞癌转录调控网络 中国 科技论文在线////0>. 联合 iTRAQ-LC MS/MS技术构建下咽鳞癌 # 转录调控网络1,3 1 2 1 1 1** 朱刚才 ,刘勇 ,蔡耿明 ,谭浩蕾 ,黄美玲 ,张欣 5 (1. 中南大学湘雅医院耳鼻咽喉头颈外科; 2. 福建医科大学附属泉州第一医院耳鼻喉头颈外科; 3. 耳鼻咽喉重大疾病湖南省重点实验室) 摘要: 目的:比较分析下咽鳞癌的全蛋白质组学信息,构建其转录调控网络。 方法 快递客服问题件处理详细方法山木方法pdf计算方法pdf华与华方法下载八字理论方法下载 :利用同 位素标记相对和绝对定量联合二维液相色谱串联质谱分析 10 例下咽鳞癌癌旁,原发部位以 10 及转移淋巴结的蛋白丰度 表 关于同志近三年现实表现材料材料类招标技术评分表图表与交易pdf视力表打印pdf用图表说话 pdf 达,筛选显著差异表达蛋白谱;通过 Metacore 软件中的转录调 控及转 录因子分析法对其进行转 录调控网络 的构建。 结果:通过差异表达谱产生 的转 录调控 网络中,统计分析最显著意义的前五位的调控网络由 48-135 个蛋白组成,p 值从 6.80e 102 到 2.44e 232。这些得分最高 的网络包括通过 SP1、C-myc、P53 等中心转 录因子。 结 论:Sp1/C-myc/P53 等中心转录因子在下咽癌的发生发展及转移的过程中可 能发挥重要作用。 15 关键词:头颈鳞癌;下咽鳞癌;蛋白质组学;iTRAQ ;Metacore ;转录调控网 络 中图分类号:R739.6 Transcriptional Regulation Network in Hypopharxngeal Squamous Carcinoma:intergrated itraq proteomics and 20 bioinformational technology 1,3 1 2 1 1 ZHU GANGCAI , Liu Yong , Cai Gengming , Tan Haolei , Huang meiLing , 1 Zhang Xin 1. Otolaryngology Department of xiangya hospital, Centeral South University, Changsha, 410008, Hunan, China.; 25 2. ENT department in The first affiliated quanzhou hospital of fujian Medical University; 3. Otolaryngology Major Disease Research Key Laboratory of Hunan Province, Changsha, 410008, Hunan, China. Abstract: Purpose:Established the transcriptional regulatory network in Hypopharyngeal squamous carcinoma. Method: Compare the alterative protein expression in 10 sublocation of 30 hypophaxygeal squamous carcinoma tissues through integrateion of Itraq with LC-MS/MS technology and Metacore software assayTranscription Regulation and Transcriptional Factor algorithm. Results: The most significant transcriptional regulatory network are genernated, including 48-135 protein, which presents powful value from 6.8*e 102 to 2.44*e 232.SP1/c-MYC/P53 are the top three hub networks in them. Conclusion: SP1/c-myc/P53 may 35 play some important role in the inination and development of Hypopharyngeal squamous carcinomaKey words: Head and Neck Squamous Carcinoma; Hypopharyngeal Squamous Carcinoma; Proteomics; iTRAQ; Metacore; Transcriptional regulatory Network 40 0 引言 下咽鳞癌是头颈鳞癌中恶性程度最高的肿瘤。尽管近年来诊治手段不断更新, 其五年生 存率并未明显改善。除其发病部位隐蔽,早期无明显自觉症状外,易早期淋巴 结转移/病程 基金项目:高等学校博士学科点专项科研基金课 题 快递公司问题件快递公司问题件货款处理关于圆的周长面积重点题型关于解方程组的题及答案关于南海问题 (No:206 作者简介:朱刚才(1988-),男,博士研究生,头颈肿瘤 通信联系人:张欣(1966年-)男,主任医师,头颈肿瘤. E-mail: xinzhang@//. - 1 - 中国 科技论文在线////. 进展快亦是下咽鳞癌预后差的原因。目前针对下咽癌鳞癌的诊疗预防中,临床上对于下 咽鳞癌的发生发展及淋巴转移的分子机制尚不清楚。本课题组拟利用同位素标记相对和绝对 45 定量isobaric tags for relative and absolute quantification, iTRAQ 联合二维液相色谱串联质谱 Two-dimensional-liquid chromatography-tandem mass spectrometry, LC-MS/MS技术 对 10 例下咽鳞癌的癌旁、原发灶以及转移的淋巴结组织进行蛋白质表达相对定量,筛选出 在下咽鳞癌的发生及转移过程中表达具有显著差异的蛋白,并对其进行转录调控网络分析。 1 材料与方法 50 1.1 临床标本与蛋白的提取 分别收集湘雅医院 2010年 2-11月住院期间 10例下咽鳞癌患者的癌旁/原发病灶/淋巴结 的新鲜组织标本,于液氮中保存。所有标本均在病理确诊之后,手术及辅助治疗之前进行。 本次实验已获得湘雅医学院伦理委员会的通过。所有病例术前均经临床病理证实, 其中 T2N2M0 4 例、T3N1M0 3 例、T3N2M01 例、T3N3M0 2 例。组织蛋白的提取与标记方法参 [1] 55 照课题组前期研究 。 1.2 iTRAQ 标记 参照 iTRAQ 试剂盒(Applied Biosystem)说明 书 关于书的成语关于读书的排比句社区图书漂流公约怎么写关于读书的小报汉书pdf ,分别标记原发部位癌组织(Ca): iTRAQ 121; 转移的淋巴结组织(N+):iTRAQ 119;癌旁组织(NAT):iTRAQ 118。 在整个试验过程中,共进行了两次平行的标记鉴定。 601.3 肽段分离与鉴定 [1] 参照课题组前期方法 。简言之,混合不同标记的样本分别被第一维强阳离子柱 (polysufoethyl column,2.1mm*100mm, 5u,200A, The Nest Group,Inc.MA); 第二维反相液湘 色谱(ZORBAX 300SB-C18 column5 ?m, 300?, 0.1 x 15 0mm , microm ,USA) 分离并脱盐 65 后,经质谱仪扫描。MS扫描范围: 400-1800;MS/MS扫描范围: 100-2 000; IDA 采集模式:一个图谱选择四个最强的母离子进行串级扫描。应用Protein pilot 4.0 软件(美国AB SCIEX 公司产品)进行对质谱结果进行肽段鉴定鉴定和定量,搜库参数定义 为(1 )样品类型:iTRAQ 8 plex (Peptide Labeled );(2 )半胱氨酸烷基化:MMTS; (3 )酶解:Trypsin;(4 )种属:Homo sapiens;(5)数据库:IPI3.45。 701.4 转录调控网络的构建 基于protein pilot软件对鉴定到的蛋白质打分, 报告 软件系统测试报告下载sgs报告如何下载关于路面塌陷情况报告535n,sgs报告怎么下载竣工报告下载 阈值为1.3, 对应蛋白质的假阳性率 为5%。软件依据同位素报告基团的相对含量进行蛋白质定量, 以NAT 癌旁组织蛋白m/z 118 为对照, 选择丰度变化1.2或0.8的蛋白作为差异蛋白。将候选差异蛋白信息输入Metacore 75 软件美国、汤森路透集团,选择转录调控及转录因子分析法对其进行转录调控网络的构建。 2 结果 2.1 蛋白质质谱的鉴定 两个 MS重复的数据相结合,鉴别出对应 853个蛋白质的 19882 条多肽,利用 Protein - 2 - 中国 科技论文在线////. Pilot 软件进行搜库, iTRAQ试剂标记质谱鉴定的蛋白和肽段的数目显示均见表 1-1 80 表 1-1 质谱鉴定 蛋白得分阈值 蛋白数目 分组前蛋 肽段 质谱鉴定 占总质谱比例(%) 可信度% 白数目 2.0 99 640 2555 18074 4092531.0 a 1.3 95 853 3263 19882 4367333.1 0.47 66 1099 4550 23046 4817836.5a:本文所后续结果采用阈值 重复的两个MS数据交叉部分占蛋白质总量的78%,其中432个蛋白覆盖至少两条肽段,通过 iTRAQ标记,符合1%的总错误发现率蛋白643个。 85 2.2 蛋白差异表达谱 下咽癌原发部位组与癌旁组织相比共有382个蛋白的丰度有变化,177个蛋白过度表达 (1.2倍以上), 205个蛋白低表达(小于0.8倍)本文仅列举排名前50位的差异蛋白(见表1-2)。 表 1-2 下咽癌原发病灶/癌旁组织中按比值排名前 50 位的上调与下调的蛋白 IPI编号 蛋白 Ca/NAT 上调 IPI00418163.3 C4B complement component 4B preproprotein 12.5892 IPI00002352.4 MYLPF Myosin regulatory light chain 2, skeletal muscle isoform 5.4450 IPI00657670.1 - Apolipoprotein C-III variant 15.3456 IPI00873767.1 FUBP1 68 kDa protein 4.5290 IPI00829626.1 IGL@ IGL@ protein 4.0926 IPI00024915.2 PRDX5 Isoform Mitochondrial of Peroxiredoxin-5,mitochondrial precursor 4.0551 IPI00030362.1 PLP2 Proteolipid protein 2 4.0551 IPI00854568.1 - PC1/MRPS28 fusion protein3.5645 IPI00018>0402.2 SH3BGRL3 Putative uncharacterized protein3.4356 IPI00384444.5 KRT14 Keratin, type I cytoskeletal 14 3.0479 IPI00450768.7 KRT17 Keratin, type I cytoskeletal 17 3.0200 IPI00304962.3 COL1A2 Collagen alpha-2I chain precursor2.8314 IPI00028444.1 PLIN Perilipin 2.8314 IPI00217465.5 HIST1H1C Histone H1.2 2.6792 IPI00473011.3 HBD;HBB Hemoglobin subunit delta 2.6546 IPI00643348.2 COMP 80 kDa protein 2.6546 IPI00029739.5 CFH Isoform 1 of Complement factor H precursor 2.5586 IPI00218245.1 EVL Isoform 1 of Ena/VASP-like protein 2.3988 IPI00219037.5 H2AFX Histone H2A.x 2.3550 IPI00296099.6 THBS1 Thrombospondin-1 precursor 2.3121 IPI00789376.1 KNG1 KNG1 protein 2.2491 IPI00654755.3 HBB Hemoglobin subunit beta2.2284 - 3 - 中国 科技论文在线////. IPI00219953.5 CMPK1 cytidine monophosphate UMP-CMP kinase 1, cytosolic 2.2284 IPI00027341.1 CAPG Macrophage-capping protein 2.1878 IPI00031461.1 GDI2 Rab GDP dissociation inhibitor beta 2.1878 IPI00003527.5 SLC9A3R1 Ezrin-radixin-moesin-binding phosphoprotein 502.1677 IPI00019580.1 PLG Plasminogen precursor 2.1478 IPI00022793.5 HADHB Trifunctional enzyme subunit beta, mitochondrial precursor 2.1086 IPI00010471.5 LCP1 Plastin-22.0701 IPI00007960.4 POSTN Isoform 1 of Periostin precursor 2.0324 IPI00784985.1 IGK@ IGK@ protein 2.0137 IPI00005969.3 CAPZA1 F-actin-capping protein subunit alpha-11.9055 IPI00792758.1 ARHGDIB 21 kDa protein 1.9055 IPI00022488.1 HPX Hemopexin precursor 1.8707 IPI00396378.3 HNRNPA2B1 Isoform B1 of Heterogeneousnuclear ribonucleoproteins A2/B11.8365 IPI00019563.3 GIMAP4 GTPase IMAP family member 4 1.8365 IPI00376199.3 IRF2BP2 Isoform 1 of Interferon regulatoryfactor 2-binding protein 2 1.8365 IPI00024664.1 USP5 Isoform Long of Ubiquitin carboxyl-terminal hydrolase 51.8197 IPI00418431.1 ASPN ASPN protein 1.8030 IPI00298828.3 APOH Beta-2-glycoprotein 1 precursor 1.8030 IPI00028414.3 GMFG Glia maturation factor gamma 1.8030 IPI00872944.1 NUCKS1 34 kDa protein 1.7701 IPI00027463.1 S100A6 Protein S100-A6 1.7701 IPI00479145.2 KRT19 Keratin, type I cytoskeletal 19 1.7378 IPI00413641.7 AKR1B1 Aldose reductase 1.7219 IPI00414554.5 ARPC5L Actin-related protein 2/3 complex subunit 5-like protein 1.7219 IPI00014516.1 CALD1 Isoform 1 of Caldesmon1.7061 IPI00790739.1 ACO2 Aconitase 2, mitochondrial1.6904 IPI00783228.3 SPTB spectrin beta isoform a 1.6904 IPI00456887.2 HNRNPUL2 Heterogeneous nuclear ribonucleoproteinU-like protein 21.6749 IPI00303300.3 FKBP10 FK506-binding protein 10 precursor1.6596 IPI00847179.1 APOA4 apolipoprotein A-IV precursor 1.6444 下调 IPI00219029.3 GOT1 Aspartate aminotransferase, cytoplasmic0.0550 IPI00027377.3 ACAN aggrecan isoform 2 precursor 0.0745 IPI00007856.1 MYH2 Myosin-20.1038 IPI00847635.1 SERPINA3 Isoform 1 of Alpha-1-antichymotrypsin precursor0.1959 IPI00216798.5 MYL2 Myosin regulatory light chain 2,ventricular/cardiac muscle isoform0.2089 IPI00009865.2 KRT10 Keratin, type I cytoskeletal 10 0.2148 IPI00872223.1 MYH7 223 kDa protein 0.2148 IPI00009866.6 KRT13 Isoform 1 of Keratin, type I cytoskeletal 13 0.2249 IPI00298497.3 FGB Fibrinogen beta chain precursor 0.2270 IPI00550069.3 RNH1 Ribonuclease inhibitor 0.2291 - 4 - 中国 科技论文在线////. IPI00029733.1 AKR1C1 Aldo-keto reductase family 1 member C1 0.2512 IPI00300502.2 MYOZ1 Myozenin-1 0.2535 IPI00423461.3 IGHA2 Putative uncharacterized proteinDKFZp686C02220 Fragment0.2729 IPI00290078.5 KRT4 keratin 40.2805 IPI00019038.1 LYZ Lysozyme C precursor 0.2911 IPI00179357.2 TTN Isoform 7 of Titin 0.3105 IPI00012119.1 DCN Isoform A of Decorin precursor 0.3311 IPI00216070.3 MYL1 Myosin light chain 1, skeletal muscle isoform 0.3373 IPI00032325.1 CSTA Cystatin-A0.3404 IPI00328753.1 KTN1 Isoform 1 of Kinectin 0.3532 IPI00305064.1 CD44 Isoform CD44 of CD44 antigen precursor0.3698 IPI00218474.5 ENO3 Beta-enolase0.3733 IPI00296922.4 LAMB2 Laminin subunit beta-2 precursor 0.3802 IPI00879810.1 SPTAN1 Uncharacterized protein SPTAN1 0.3837 IPI00291410.3 C20orf114 Isoform 1 of Long palate,lung and nasal epithelium carcinoma-associated protein 1 precursor 0.3873 IPI00021700.3 PCNA Proliferating cell nuclear antigen0.3873 IPI00514424.2 PPT1 Palmitoyl-protein thioesterase 1 0.3873 IPI00290077.1 KRT15 Keratin, type I cytoskeletal 15 0.4018 IPI00394838.2 ACLY ATP citrate lyase isoform 2 0.4018 IPI00008580.1 SLPI Antileukoproteinase precursor 0.4018 IPI00082931.1 SPRR3 Small proline-rich protein 3 0.4055 IPI00221234.7 ALDH7A1 aldehyde dehydrogenase 7 family, member A1 0.4055 IPI00025465.1 OGN Mimecan precursor 0.4093 IPI00032826.1 ST13 Hsc70-interacting protein0.4169 IPI00179330.6 UBC;UBB;RPS27A ubiquitin and ribosomal protein S27a precursor 0.4207 IPI00013991.1 TPM2 Isoform 1 of Tropomyosin beta chain 0.4207 IPI00215780.5 RPS19 40S ribosomal protein S190.4285 IPI00178926.2 IGJ immunoglobulin J chain 0.4571 IPI00478410.2 ATP5C1 Isoform Liver of ATP synthase subunit gamma,mitochondrial precursor 0.4571 IPI00744889.2 CDH1 E-cadherin0.4571 IPI00219221.3 LGALS7;LOC728910;GAL7 Galectin-7 0.4613 IPI00409717.1 EIF4A2 Isoform 2 of Eukaryotic initiation factor 4A-II 0.4613 IPI00640817.1 AK1 Adenylate kinase 1 0.4613 IPI00877703.1 FGG 52 kDa protein 0.4656 IPI00643435.1 ATAD3A Isoform 1 of ATPase family AAAdomain-containing protein 3A0.4656 IPI00719669.4 MRLC2 Myosin regulatory light chain 0.4699 IPI00514330.5 HDGF hepatoma-derived growth factor isoform c0.4742 IPI00027223.2 IDH1 Isocitrate dehydrogenase [NADP] cytoplasmic 0.4786 IPI00025874.2 RPN1 Dolichyl-diphosphooligosaccharide--proteinglycosyltransferase 67 kDa subunit precursor0.4786 - 5 - 中国 科技论文在线////. IPI00217966.7 LDHA Isoform 1 of L-lactate dehydrogenase A chain 0.483190在转移的淋巴结组与癌旁组织组相比共有386个蛋白的丰度有变 化,177个蛋白过度表 达(1.2倍以上),209个蛋白低表达(小于0.8倍)(详见表1-3)。 表 1-3 转移的淋巴癌组织/癌旁组织中按比值排名前 50 位的上调与下调 的蛋白 IPI编号 蛋白N+/NAT 上调 IPI00297056.2 CRNN Cornulin 28.5759 IPI00002352.4 MYLPF Myosin regulatory light chain 2, skeletal muscle isoform 28.3139 IPI00216070.3 MYL1 Myosin light chain 1, skeletal muscle isoform 20.7014 IPI00243742.7 MYL3 Myosin light chain 318.1970 IPI00009866.6 KRT13 Isoform 1 of Keratin, type I cytoskeletal 13 14.3219 IPI00290078.5 KRT4 keratin 4 13.3045 IPI00418163.3 C4B complement component 4B preproprotein 12.5892 IPI00465084.6 DES Desmin 8.9536 IPI00216798.5 MYL2 Myosin regulatory light chain 2,ventricular/cardiac muscle isoform8.7096 IPI00013991.1 TPM2 Isoform 1 of Tropomyosin beta chain 8.1658 IPI00007856.1 MYH2 Myosin-27.9433 IPI00872223.1 MYH7 223 kDa protein 7.8705 IPI00657670.1 - Apolipoprotein C-III variant 17.4473 IPI00019884.1 ACTN2 Alpha-actinin-2 6.8549 IPI00473011.3 HBD;HBB Hemoglobin subunit delta 6.6069 IPI00654755.3 HBB Hemoglobin subunit beta6.5464 IPI00216983.6 CA3 Carbonic anhydrase 3 6.0256 IPI00829626.1 IGL@ IGL@ protein 5.6494 IPI00816618.1 HBG2 Hemoglobin gamma-G Fragment 5.4954 IPI00217465.5 HIST1H1C Histone H1.2 5.3456 IPI00873767.1 FUBP1 68 kDa protein 5.0119 IPI00844276.1 LAMA2 laminin alpha 2 subunit isoform a precursor 4.9659 IPI00178352.5 FLNC Isoform 1 of Filamin-C4.6132 IPI00300502.2 MYOZ1 Myozenin-1 4.6132 IPI00032164.1 CSRP3 Cysteine and glycine-rich protein 3 4.5709 IPI00025879.1 MYH1 Myosin-14.2462 IPI00640817.1 AK1 Adenylate kinase 1 4.0551 IPI00410714.5 HBA1;HBA2 Hemoglobin subunit alpha 4.0179 IPI00218474.5 ENO3 Beta-enolase3.9084 IPI00410261.3 MYBPC1 myosin binding protein C, slow type isoform 1 3.6983 IPI00215983.3 CA1 Carbonic anhydrase 1 3.2810 IPI00003527.5 SLC9A3R1 Ezrin-radixin-moesin-binding phosphoprotein 503.2509 IPI00219037.5 H2AFX Histone H2A.x 3.0761 IPI00418431.1 ASPN ASPN protein 3.0479 IPI00290077.1 KRT15 Keratin, type I cytoskeletal 15 2.8840 - 6 - 中国 科技论文在线////. IPI00604785.1 MCTS1 MCTS1 protein 2.8840 IPI00218245.1 EVL Isoform 1 of Ena/VASP-like protein 2.8054 IPI00295741.4 CTSB Cathepsin B precursor 2.7797 IPI00304962.3 COL1A2 Collagen alpha-2I chain precursor2.6792 IPI00895929.1 TNXB tenascin XB isoform 1 precursor 2.6546 IPI00027350.3 PRDX2 Peroxiredoxin-2 2.5586 IPI00010402.2 SH3BGRL3 Putative uncharacterized protein2.5119 IPI00010471.5 LCP1 Plastin-22.4889 IPI00218848.5 ATP5I ATP synthase, H+ transporting, mitochondrial F0 complex,subunit E 2.3988 IPI00021304.1 KRT2 Keratin, type II cytoskeletal 2 epidermal2.3768 IPI00028031.2 ACADVL Isoform 1 of Very long-chain specificacyl-CoA dehydrogenase, mitochondrial precursor 2.3335 IPI00005537.2 MRPL12 39S ribosomal protein L12, mitochondrial precursor2.2284 IPI00789376.1 KNG1 KNG1 protein 2.2284 IPI00291005.8 MDH1 Malate dehydrogenase, cytoplasmic 2.2080 IPI00298281.3 LAMC1 Laminin subunit gamma-1 precursor2.1677 下调 IPI00027377.3 ACAN aggrecan isoform 2 precursor 0.0433 IPI00219221.3 LGALS7;LOC728910;GAL7 Galectin-7 0.0705 IPI00450768.7 KRT17 Keratin, type I cytoskeletal 17 0.0731 IPI00009865.2 KRT10 Keratin, type I cytoskeletal 10 0.0855 IPI00291410.3 C20orf114 Isoform 1 of Long palate,lung and nasal epithelium carcinoma-associated protein 1 precursor 0.1294 IPI00071509.1 PKP1 Isoform 2 of Plakophilin-10.1318 IPI00007797.3 FABP5;FABP5L7 Fatty acid-binding protein, epidermal 0.1419 IPI00217963.3 KRT16 Keratin, type I cytoskeletal 16 0.1542 IPI00019038.1 LYZ Lysozyme C precursor 0.1600 IPI00029733.1 AKR1C1 Aldo-keto reductase family 1 member C1 0.1629 IPI00007960.4 POSTN Isoform 1 of Periostin precursor 0.1905 IPI00010951.2 EPPK1 Epiplakin0.1995 IPI00384444.5 KRT14 Keratin, type I cytoskeletal 14 0.2089 IPI00872047.1 SMU1 Uncharacterized protein SMU1 Fragment 0.2512 IPI00013933.2 DSP Isoform DPI of Desmoplakin0.2559 IPI00554788.5 KRT18 Keratin, type I cytoskeletal 18 0.2559 IPI00167949.6 MX1 Interferon-induced GTP-binding protein Mx1 0.2559 IPI00016610.2 PCBP1 PolyrC-binding protein 10.2582 IPI00879531.1 - 9 kDa protein0.2606 IPI00304903.4 SPRR1B Cornifin-B 0.2630 IPI00293665.8 KRT6B Keratin, type II cytoskeletal 6B 0.2780 IPI00021536.1 CALML5 Calmodulin-like protein 5 0.2805 IPI00848342.1 LTF Lactotransferrin precursor0.2858 IPI00011654.2 TUBB Tubulin beta chain 0.2911 IPI00789324.1 JUP JUP protein0.3133 IPI00019329.1 DYNLL1 Dynein light chain 1, cytoplasmic 0.3133 - 7 - 中国 科技论文在线////. IPI00031549.5 DSC3 Isoform 3A of Desmocollin-3 precursor0.3251 IPI00022204.2 SERPINB3 Isoform 1 of Serpin B3 0.3311 IPI00029510.1 LY6D Lymphocyte antigen 6D precursor 0.3311 IPI00549725.6 PGAM1 Phosphoglycerate mutase 1 0.3342 IPI00032140.4 SERPINH1 Serpin H1 precursor0.3404 IPI00854806.1 IGKV1-5 IGKV1-5 protein 0.3404 IPI00298497.3 FGB Fibrinogen beta chain precursor 0.3532 IPI00028444.1 PLIN Perilipin 0.3532 IPI00867588.1 FN1 Isoform 13 of Fibronectin precursor 0.3597 IPI00479366.2 - Uncharacterized protein ENSP00000351543 Fragment 0.3664 IPI00004358.4 PYGB Glycogen phosphorylase, brain form 0.3733 IPI00018219.1 TGFBI Transforming growth factor-beta-inducedprotein ig-h3 precursor 0.3767 IPI00219468.4 PFN2 Isoform IIa of Profilin-20.3981 IPI00005490.1 GOLPH3 Golgi phosphoprotein 30.3981 IPI00337392.1 C19orf33 Isoform 2 of Immortalization up-regulated protein0.4018 IPI00031547.1 DSG3 Desmoglein-3 precursor0.4130 IPI00015117.2 LAMC2 Isoform Long of Laminin subunit gamma-2 precursor0.4169 IPI00014516.1 CALD1 Isoform 1 of Caldesmon0.4285 IPI00218918.5 ANXA1 Annexin A1 0.4325 IPI00514330.5 HDGF hepatoma-derived growth factor isoform c0.4325 IPI00009009.4 CWC15 Protein CWC15 homolog0.4325 IPI00219301.7 MARCKS Myristoylated alanine-rich C-kinase substrate 0.4529 IPI00383581.3 GANAB Isoform 1 of Neutral alpha-glucosidase AB precursor0.4529 IPI00026230.1 HNRNPH2 Heterogeneous nuclear ribonucleoprotein H2 0.45712.3 转录调控网络的构建 95 为了进一步探索参与转移的未知的通路及其之间的关系,上调和下调的 蛋白质被提交到 MetaCore,由 Transcription Regulation and Transcriptional Factor algorithm的 Analyze Networks (AN)计算,。将差异蛋白表达谱输入 Metacore 软件。通过输入差异蛋白产 生一转录调控 102 232 网络,p 值排列前五位的调控网络由 48-135 个蛋白组成,p 值从 6.80e 到 2.44e 。这些 得分最高的网络包括通过 SP1、c-myc、p53 等转录因子的激活(图 1、2、3)。 - 8 - 中国 科技论文在线////. 100图 1: Metacore 分析的蛋白质排名第一位的转录调控网络: 以 SP1 为中心的转录调控网络; 以红色代表蛋白上调,蓝色代表蛋白下调。 - 9 - 中国 科技论文在线////. 图 2: Metacore 分析的蛋白质排名第二位的转录调控网络:以 c-myc 为 中心的转录调控网 105 络;以红色代表蛋白上调,蓝色代表蛋白下调。 - 10 - 中国 科技论文在线////. 图 3: Metacore 分析的蛋白质排名第三位的转录调控网络: 以 p53 为中心的转录调控网络; 以红色代表蛋白上调,蓝色代表蛋白下调。 1103 讨论 iTRAQ 技术其具有能同时标记多重样品、灵敏度高、能标记几乎所有的肽段的优势, 而且可与液相质谱联合应用,二维液相串联质谱(2D LC-MS/MS)已经成为目 前蛋白质鉴 定和定量的重要方法之一,与传统的双相凝胶电泳和双相差异凝胶电泳相比, 2D LC-MS/MS 115 能够获得更多的蛋白定性和定量的信息,包括对膜蛋白、疏水性蛋白或大分子蛋白和低丰度 [2] 蛋白的检测 。本文利用iTRAQ标记10例下咽鳞癌的癌旁组织、原发病灶及转移的淋巴病变; 联合2D LC-MS/MS分析比较它们的全蛋白组学信息,鉴定出853个蛋白丰度表达变化。这从 另外的角度说明了iTRAQ技术的高灵敏度。 Metacore软件基于的底层数据库是世界上唯一一个完全通过人工阅读全文文献和实验 [3] 120 数据后进行整合的高准确率的系统生物学数据库 。本研究中转录调控网络的构建正基于 Metacore软件人工修正后的数据库,蛋白之间的互相调控信息都有相关文献信息支持。本研 究表明Sp1、C-myc、P53为中心的调控网络可能在下咽鳞癌的发生发展中发挥重要的作用。 [4] SP1是一类具有C2H2锌指蛋白,可调控启动子中GC盒子的转录因子 。越来 越多的研 [5, 6] 究表明Sp1蛋白在包含鳞癌在内多种肿瘤组织的上调激活 ,并在肿瘤的发生发展以及侵袭 - 11 - 中国 科技论文在线////. 125 转移中发挥作用。然而目前,针对SP1蛋白在下咽鳞癌的研究却并不多见。我们之前的研究 提示上皮间质转换和凋亡逃避是转移性头颈鳞癌的两大特性。在本文构建的SP1调控网络 中,Sp1可调控可能参与上述过程中的重要分子比如:PCNA, β-catenin, 纤维素蛋白。 P53蛋白为中心的转录调控网