基于iTRAQ技术的结直肠癌蛋白标志物鉴定
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摘要
背景与目的
结直肠癌是全世界范围内发病率最高的恶性肿瘤疾病之一。据世界卫生组织（WHO）资料显示，结直肠癌居男性常见恶性肿瘤发病率第三位，居女性常见恶性肿瘤发病率第二位，2008年度全世界估计总新增病例超过120万例，居第2位，死亡病例超过608,700人，居第4位。结直肠癌发病率具有明显地域差异。其中高发地区为澳大利亚、新西兰、欧洲和北美，非洲和中南亚为低发地区。世界范围内发病率男女之比约为1.16：1。
随着我国人民生活水平的不断提高和改善，以及饮食习惯的变化，我国结直肠癌发病率及死亡率亦逐年升高，大中城市尤其明显，资料表明，中国大陆13.4亿人口，结直肠癌在全国肿瘤登记地区恶性肿瘤发病率居第三位，发病39.7万例，发病率29.62/10万，居男性发病率第四位，女性发病率第三位；结直肠癌死亡率居第五位，死亡19.0万例，死亡率14.15/10万，居男性死亡率第五位，女性死亡率第四位。
多项研究显示，与结直肠癌发生相关的危险因素包括吸烟、缺乏体育锻炼、超重及肥胖、红色及加工处理的肉类消费和过度饮酒。结直肠癌的发生和发展是一个历时数十年，多阶段进展，多因素参与，伴随一系列的分子事件的复杂进程。多年的研究揭示了大量的发生于DNA和RNA水平的分子特征性改变，形成了人们现在对结直肠癌发生的分子机制的认识,包括了基因组DNA序列的突变，DNA拷贝数的改变，启动子超甲基化，miRNA表达水平改变等，最终这些变化影响了结直肠癌蛋白质表达谱的改变。
影响结直肠癌预后的最重要因素就是早期诊断与早期治疗，研究显示TNM I期患者的五年生存率可达90%以上，而有远处器官转移的IV期患者5年生存率只有大约10%。虽然经过多年的基础和临床共同努力，结直肠癌死亡率和生存周期并未获得明显改善和延长，究其原因，在于相当一部分患者在就诊时就已经处于肿瘤进展期。有报道表明，30-40%的就诊患者已经存在区域侵润和转移性疾病，并无法通过单纯的手术治疗获得治愈，并且超过一半的患者术后复发并且罹难于肿瘤复发和转移。研究表明，通过对广泛人群进行筛查的结直肠癌早期检测和诊断，以及对早期结直肠癌进行干预，可以有效的延长生存期，而这一切依赖于高效而准确的结直肠癌诊断和预后生物标记物的发现及应用。
蛋白质参与了人类生理、病理过程的绝大部分活动，在疾病发生、机体反应和康复的各个进程都发挥着重要的作用，因此，作为生命活动的重要作用分子形式，蛋白质标志物自然是生物标志物筛选与鉴定的焦点。
iTRAQ技术是由美国ABI应用生物系统公司（Applied BiosystemsIncorporation, ABI)开发的同位素标记相对和绝对定量技术(isobaric tags forrelative and absolute quantitation, iTRAQ)的简称，相对于传统的定量鉴定，具有无可比拟的优点：在同一实验中，最多可以对多达4到8种不同样本进行蛋白定量比较，可以对样本中的绝大部分蛋白质进行标记，比如可以对磷酸化蛋白、糖基化蛋白等翻译后修饰蛋白进行定量、定性研究，从中获得更为详尽的样品信息，标记过程更简便，在室温条件下1小时即可完成，质谱检测更为灵敏。
本课题研究采用的蛋白组学研究策略是以同位素相对绝对定量技术（iTRAQ）为基础，通过联合多维固/液相色谱技术与串联质谱技术分离和鉴定蛋白质，对比结直肠癌与配对的正常对照粘膜组织蛋白质组定量结果和同一患者术前与术后血清蛋白质组定量结果，分别描绘结直肠癌患者组织对比和血清对比差异蛋白质谱，通过比对组织和血清的差异蛋白质谱，挑选一组在组织和血清中均有一致性差异表达的差异蛋白，经生物信息学分析后行Wester blot、免疫组化验证，筛选出一组具有理想诊断和/或预后价值或可作为治疗靶点的免疫组织化学标志物，以期提高结直肠癌疾病的早期诊断水平、改善患者预后和/或为治疗提供可能的靶点。相比于以往的研究，本课题的创新性体现在在一次iTRAQ标记实验中同时比对同一患者的组织与血清差异表达蛋白谱，筛选在组织学和血清学水平具有一致性差异的蛋白标志物，增强了后期验证结果的可靠性。
方法
本研究收集1例结直肠癌患者癌组织和配对正常粘膜组织，以及同一患者的术前和术后血清，经蛋白提取和经胰酶消化为多肽后，分别以同位素相对与绝对定量试剂盒（iTRAQ）标记组织与血清蛋白肽段，将二者混合后经过强阳离子交换柱（SCX）和反相色谱柱（RPLC）的二维分离，之后联用MALDI质谱技术对分离后的样品蛋白质多肽质谱图进行定量鉴定，经Mascot数据库检索，描绘结直肠癌患者组织与血清差异蛋白质谱。挑选出感兴趣的目的蛋白后，登录GeneOntcology与Swiss-Prot数据库进行差异蛋白质的亚细胞结构定位和功能分析。
经生物信息学分析，挑选出差异表达蛋白标志物，经Western blot和免疫组织化学法验证其表达水平。收集除用于iTRAQ定量的结直肠癌病例以外的3例与之临床病理特征匹配的结直肠癌病例，共4例的结直肠癌肿瘤组织及配对正常组织，经前述冰冻组织保存及蛋白提取方法，进行蛋白提取、定量后，分别取总蛋白各20μg进行Western blot验证，以PD QUEST软件半定量分析。收集80例结直肠癌患者肿瘤组织和匹配的正常对照粘膜组织，经福尔马林固定石蜡包埋处理后切片，经逐步脱蜡至水、双氧水（H2O2）封闭内源酶活性、高温高压抗原修复、10%牛血清封闭、抗原特异性一抗过夜孵育、漂洗后种属特异二抗常温孵育、DAB显色及苏木素复染后，由两位病理医师分别对免疫组化（IHC）结果进行双盲评分。IHC评分标准：在5-10个随机视野观察200个上皮细胞，从免疫阳性染色细胞比例和免疫染色强度两个方面进行综合评分，两位医师的评分平均后作为最终结果。根据免疫阳性细胞比例不同，分为：0分，阴性；1分，<10%阳性；2分，11%-50%阳性；3分，≥51%阳性。根据免疫染色强度不同，分为：0分，阴性；1分，弱阳性；2分，阳性；3分，强阳性。为系统评价蛋白标志物Prenylcysteine oxidase1和Peroxiredoxin-4的诊断和/或预后价值。为评价筛选的蛋白标志物的诊断可靠性，以免疫组化评分为依据，从低到高设立7个诊断阈值，分别是评分≥0、1、2、3、4、5和6。统计分析在不同的诊断阈值下，蛋白标志物用于结直肠癌（CRC）诊断的特异度、灵敏度、阴性预测值与阳性预测值、约登指数（Youden Index）和一致性检验Kappa值，绘制受试者工作特征曲线（ROC），确定界值点，计算ROC曲线下面积AUC。
结果
以同位素相对与绝对定量（iTRAQ）为基础、固/液相二维分离及MALDI质谱技术描绘结直肠癌组织与血清差异蛋白谱，经Scaffold筛选，NCBI_human库检索，共鉴定到血清蛋白198个,其中差异蛋白102个；肿瘤组织鉴定到蛋白568个，其中差异蛋白391个。经过比对发现，共有13个蛋白在血清和组织中都鉴定为差异表达蛋白，其中5个蛋白在肿瘤组织中表达水平上调，8个在肿瘤组织中表达水平下调，10个蛋白在术前血清中表达水平上调，3个蛋白在术后2周血清表达水平上调,4个蛋白表达差异具有一致性（我们规定，在肿瘤组织表达升高/降低和在术前血清表达升高/降低，称为表达差异具有一致性）。通过检索UniProt knowledgebase数据库(Swiss-Prot/TrEMBL, www.expasy.org)和GeneOntology (GO) Database数据库(http://www.geneontology.org/)，我们对筛选出的13个差异蛋白质的生物学功能进行了GO分析。GO分析结果显示此13个差异蛋白质，大部分（84.6﹪）具有两个或两个以上的分子功能或参与的生物学进程，其中与结合（Binding）功能有关的占84.6﹪，此外，还与生物功能调节（76.9%），细胞进程（69.2%），代谢进程（69.2%）和应激（84.6%）功能相关。我们选择了Prenylcysteine oxidase1， Peroxiredoxin-4，Lumican和Complement factor B这四个具有一致性差异表达的蛋白标志物进行Western-Blot实验，结果证实Prenylcysteine oxidase1， Peroxiredoxin-4这2个蛋白的表达量在肿瘤组织中高于正常组织。随后对这2个蛋白进行免疫组化验证，利用SPSS18.0软（卡方检验与Wilcoxon signed ranks检验）对免疫组化评分进行统计学分析。结果表明：与配对的正常对照组织相比，Prenylcysteine oxidase1和Peroxiredoxin-4在肿瘤组织的上调表达具有统计学意义（二者均P<0.01），二者分别在85%和71.25%的结直肠癌组织上调表达，卡方检验提示Peroxiredoxin-4的上调表达与淋巴结侵袭和TNM分期相关（P<0.05），Prenylcysteine oxidase1的上调表达与病例临床特征如年龄、性别、肿瘤部位、分化程度、肿瘤TNM分期和淋巴结侵袭之间并无显著相关性。经Logistic回归分析病例临床病理特征和蛋白标志物在肿瘤组织和正常组织的差异表达程度相关性，提示Prenylcysteine oxidase1和Peroxiredoxin-4在肿瘤组织和正常组织的差异表达程度均与淋巴结侵袭相关，OR值分别为3.386（95%可信区间，1.474-8.243）和2.858（95%可信区间，1.219-6.701）。Prenylcysteine oxidase1主要表达在胞浆，其阳性表达在95%的结直肠癌组织和60%的正常对照组织中被检测到。Peroxiredoxin-4的表达分布于胞浆和部分细胞外间质区域，其阳性表达可以在96.25%的结直肠癌组织和80%的对照正常组织样本中检测到。
Prenylcysteine oxidase1诊断结直肠癌（CRC）的诊断阈值是免疫组化评分≥3，经统计分析其CRC诊断特异度为93.75℅，敏感度为76.25℅，阳性预测值为92.4℅，阴性预测值为79.8℅，在该诊断阈值下，计算约登指数（YI）为70，收受者特征曲线（ROC）的曲线下面积（AUC）为89.4%。Peroxiredoxin-4诊断结直肠癌（CRC）的诊断阈值是免疫组化评分≥4，经统计分析其CRC诊断特异度为96.3℅，敏感度为63.8℅，阳性预测值为94.4℅，阴性预测值为72.6℅，在该诊断阈值下，约登指数为60.1，收受者特征曲线（ROC）的曲线下面积（AUC）为84.2%。
结论
我们初步描绘了结直肠癌组织与血清差异表达蛋白质谱，筛选出了一组具有较好诊断价值的蛋白标志物，验证了Prenylcysteine oxidase1和Peroxiredoxin-4在癌组织中的上调表达，为结直肠癌发生发展机制及肿瘤标志物的研究提供了新的线索。其中Prenylcysteine oxidase1对结直肠癌患者早期诊断以及Peroxiredoxin-4对结直肠癌转移相关的预后分析具有较大的价值。
Background and Aims
Colorectal cancer(CRC) is the third most commonly diagnosed cancer in males andthe second in females, with over1.2million new reported cases and608,700deathcases estimated to have occurred in2008according to WHO report. Incidence rateswere unbalanced distributed worldwide.The highest incidence rates are found inAustralia and New Zealand, Europe, and North America,whereas the lowest rates arefound in Africa and South-Central Asia. Rates are substantially higher in males thanin females.
In China the incidence and mortality of CRC is rapidly increasing in recent years,especially in big cities,mainly due to the raising of people’s living standards andchanging of people’s diets.Datas showed CRC is the third most commonly diagnosedcancer in the whole population of over1.3billion, the fourth in males and the third infemales, with over0.39million new cancer cases and0.19million deaths estimated tohave occurred in2009.
Several researches showed modifiable risk factors for colorectal cancer includesmoking, physical inactivity, overweight and obesity, red and processed meatconsumption, and excessive alcohol consumption. CRC develops through a complexprogressions of genetic and epigenetic abnormalities that transform normal epitheliuminto dysplastic epithelium, which may ultimately progress into invasive and metastaticdisease. Extensive molecular characterization of colon tumors at the DNA and RNAlevel has shaped our current knowledge about the molecular mechanisms thatinvolved in tumorigenesis,including DNA sequence mutations, DNA copy numberchanges, promoter hypermethylation, altered miRNA expression and so on, whichultimately affecting the spectrum of proteins expressed in CRC.
Despite of the advances in surgery-based comprehensive treatments for CRC, theoverall outcomes and survival terms of patients have not significantly improved or prolonged over the last few decades sinace most of the patients are diagnosed atadvanced stages.Therefore,diagnosis at early stage is considered as a crucial andpromising approach to reduce mortality and improve the prognosis.Reports haveshowed that the prognosis of CRC patients range dramaticly with five year survivalrates of more than90%for localized CRC (stage I) and only about10%for CRC thatmetastasized to distant organs (stage IV).Although considerable attentions and effortshave been spend on CRC, the reduced incidences of CRC and/or prolonged survivalterms of CRC patients were not gained during recent years, mostly due to the fact thatmost CRC patients were diagnosed at advanced stages. In fact,30–40%of patientshave regionally advanced or metastatic disease on presentation, which cannot becured by surgery alone. In addition, more than half of the patients develop recurrenceand die of the disease.It’s widely recommended to screen CRC in population as wellas interference at early stages,by which the survival terms of CRC patients could beprolonged.All these screening and early stages interference attempts are based onidentification of protein biomarkers with superior diagnosis effects.
Although biomarkers can adopt many molecular forms, proteins are major players indisease, response and recovery. Therefore, many efforts have been made to identifyprotein biomarkers.
ITRAQ is short for isobaric tags for relative and absolute quantitation,developed byApplied Biosystems Incorporation of USA. The iTRAQ is based on differentialisotopic labeling of proteins or peptides which are derived from two cell states (e.g.,healthy and tumor cells) with either light or heavy tags. This technique could label4components to the most in identification experiments simultaneously and had shownhuge advantages in throughput,sensitivity and quantitative precision to overcomesome drawbacks of traditional proteomic techniques and represented a powerfulalternative.
In the present work, iTRAQ was combined with two-dimensional liquidchromatography (2D-LC) and MS (Qstar-ESI, Applied Biosystems) to identifypotential biomarkers for CRC by two steps of comparison of protein profiles.First,weseperately compared protein profiles of CRC tissues and paired normal mucosal aswell as those of pre-surgical serum and post-surgical serum.Then the proteinsdifferentially quantificated in both tissue and serum sections were sorted out forfurther verification. The disregulated proteins were verified further by western-blot and immunohistochemistry（IHC）. The present study not only evaluated thesensitivity and specificity of the new potential biomarkers but also analysis thecorrelation and regression of the patients' clinical characteristic and the new potentialbiomarkers.Our work compared the disregulated proteins of CRC tissues and serumsto sort the protein biomarkers with accordant differential expression,this stragetycontributes to the reliablity of our results.
Methods
Tissue and serum specimens for iTRAQ were obtained from the same patient whounderwent colon cancer surgery in our hospital. CRC tissue and paired normalmucosal tissue(located at least5cm away from the tumor margin) were obtainedduring surgical resection. After excision, sample tissues were flash frozen at80°Cand stored until use.Serum samples were obtained1day before surgery and twoweeks after surgery. Peripheral venous blood samples(10ml) were taken byvenipuncture and allowed to clot.Then the serums were collected and stored at-80℃until use.
The iTRAQ labeled samples were fractionated by a SCX column and RP-LC followedby ESI-MS/MS analysis.Protein identification and quantification for the iTRAQexperiment were performed with Mascot version2.3.01(Matrix Science Inc,USA).The Scaffold version3.0(Proteome Software Inc,Portland) in Mascot software wasused for peptide identification and isoform specific quantification.
The sub-cellular location and function of the identified proteins were elucidated bythe UniProt knowledgebase (Swiss-Prot/TrEMBL,www.expasy.org) and the GeneOntology(GO) database(http://www.geneontology.org/).CRC tissue and paired normalmucosal tissue samples used in iTRAQ exprement and other3cases of CRC tissuesand paired normal mucosal tissues containing20μg of total proteins were tested bywestern blot.Two proteins from the biomarker panel analysis were selected for IHCverification using an independent and larger sample set(80cases).Immunopositivestaining was evaluated in five areas.Sections were scored as positive if epithelial cellsshowed immunopositivity in the cytoplasm, plasma membrane, and/or nucleus whenjudged independently by two scorers who were blinded to the clinical outcome.Aquantitative score was performed by estimating the percentage of immuno-positivestained cells:0, negative;1,<10%positive cells;2,11%to50%positive cells;3,≥51%positive cells. Meanwhile, the intensity of staining was scored by evaluating the average staining intensity of the positive cells (0, negative;1, weak;2, moderate; and3, strong). Finally, a total score(ranging from0to6) was obtained by adding thequantitative score and the intensity score for each of the160sections.Statisticalanalyses were carried out with SPSS19.0software (SPSS, Chicago, IL, USA).Wilcoxon signed-rank test was used to compare qualitative variables of IHC resultsbetween CRC tissues and paired normal mucosal tissues. Pearson χ2or correctionalχ2tests were used to analyze the correlation between the differential expression ofindicated proteins and patients’ clinicopathologic characteristics.Logistic regressionwas used to analysis risk factors that could effect expressions of potential proteinbiomarkers. Receiver operating characteristic (ROC) curves were used to determinethe diagnostic values of the markers.
Results
iTRAQ agents were used in the present study to label digested peptides for comparingprotein expressions between tissues of CRC and matched normal mucosal as well asthat between serums of pre-and post-surgical. Here, up-or down-regulation is statedas protein expression in CRC tissues relative to paired normal mucosa or inpre-surgical serums relative to post-surgical serums.A total of568proteins(391differentially expressed) were identified in tissues and198proteins(102differentiallyexpressed) in serums. Using the cut-off value of1.5-fold for up-regulation anddown-regulation, a total of187proteins(73up-regulated and114down-regulated) and44proteins(19up-regulated and25down-regulated) were identified as differentiallyexpressed in tissues and serums,seperately.13proteins differentially expressed in bothtissue and serum sections were sorted out for further analysis.
UniProt knowledge base (Swiss-Prot/TrEMBL) and the Gene Ontology (GO)database annotated that10of the13differential proteins were located in more thanone cellular component or organelles. In brief,38.5%,7.7%,69.2%,69.2%,23.1%,53.8%and53.8%of the13differential proteins were located inthe organelle, organelle part,extracellular region,extracellular regionpart,macromolecular complex, cell and cell part,respectively. We analyzed the proteinfunction annotated by the UniProt knowledgebase (Swiss-Prot/TrEMBL) and the GOdatabase.The following biological processes and molecular functions were involvedmore frequently:binding (84.6%), biological regulation(76.9%), cellular process(69.2%), metabolic process(69.2%), and response to stimulus (84.6%). In total,84.6% differential proteins were involved in more than one molecular function andbiological process.
To validate our proteomic results, we selected4proteins, with the accordantdifferential expression of tissues and serums,out of13differentially expressedproteins and directly assessed their levels of expression by westernblot.Prenylcysteine oxidase1and peroxiredoxin-4were validated to be up-regulatedin4CRC cases.Immunohistochemical verifications of prenylcysteine oxidase1andperoxiredoxin-4were tested in80cases of primary human CRC tissues and theirpaired normal mucosa.The scores of prenylcysteine oxidase1and peroxiredoxin-4between CRC tissues and normal mucosa show statistically significant differences.(Wilcoxon signed-ranks test, P<0.001for prenylcysteine oxidase1, P<0.001forperoxiredoxin-4). Compared with normal cells, prenylcysteine oxidase1andperoxiredoxin-4were verified to be up-regulated in85%and71.25%of the CRCcases, respectively. As the results of Pearson χ2or correctional χ2tests, theup-regulated expression of peroxiredoxin-4was found to be correlated with lymphnodes invasions and TNM stages.Interestingly, logistic correlation tests showed thatdifferential expression of both prenylcysteine oxidase1and peroxiredoxin-4betweenCRCs and normal mucosa samples were correlated with lymph nodes invasions withOR of3.386(95%Wald Confidence Limits,1.474-8.243) and2.858(95%WaldConfidence Limits,1.219-6.701),respectively.Prenylcysteine oxidase1expressionshowed cytoplasm expression,which was detected in95%of CRC samples and60%of paired normal mucosa samples.Peroxiredoxin-4showed cytoplasm andextracellular expression,which was detected in96.25%of the CRC samples and80%of matched normal mucosa tissues.Cut-off values for prenylcysteine oxidase1andperoxiredoxin-4were stastically analyzed and determined as score3and4,respectively, to distinguish CRC from paired normal mucosa by comparing the sumsof sensitivity and specificity of various diagnosis criterias; these criterias were set as I,II, III, IV, V, VI and VII (scores≥0,1,2,3,4,5and6,respectively). Relateddiagnostic values for CRC detection using prenylcysteine oxidase1andperoxiredoxin-4were shown in Table4. The areas under the curve (AUC) of thereceiver operator characteristic (ROC) curves of prenylcysteine oxidase1andperoxiredoxin-4were89.4and84.2, respectively. These results showed moderatediagnostic accuracies for prenylcysteine oxidase1and peroxiredoxin-4.
Conclusions
We performed the iTRAQ-based CRC proteomic research and identified a total of391differentially expressed proteins in tissues and102differentially expressedproteins in serums. Prenylcysteine oxidase1and peroxiredoxin-4were validated to beup-regulated in CRC compared with paired normal mucosa by western blot andimmunohistochemistry. Our results suggest that prenylcysteine oxidase1andperoxiredoxin-4are potential diagnostic or prognosis biomarkers for CRCs, and theymay play an important role in tumorigenesis and development of CRCs.
结直肠癌是全世界范围内发病率最高的恶性肿瘤疾病之一。据世界卫生组织（WHO）资料显示，结直肠癌居男性常见恶性肿瘤发病率第三位，居女性常见恶性肿瘤发病率第二位，2008年度全世界估计总新增病例超过120万例，居第2位，死亡病例超过608,700人，居第4位。结直肠癌发病率具有明显地域差异。其中高发地区为澳大利亚、新西兰、欧洲和北美，非洲和中南亚为低发地区。世界范围内发病率男女之比约为1.16：1。
随着我国人民生活水平的不断提高和改善，以及饮食习惯的变化，我国结直肠癌发病率及死亡率亦逐年升高，大中城市尤其明显，资料表明，中国大陆13.4亿人口，结直肠癌在全国肿瘤登记地区恶性肿瘤发病率居第三位，发病39.7万例，发病率29.62/10万，居男性发病率第四位，女性发病率第三位；结直肠癌死亡率居第五位，死亡19.0万例，死亡率14.15/10万，居男性死亡率第五位，女性死亡率第四位。
多项研究显示，与结直肠癌发生相关的危险因素包括吸烟、缺乏体育锻炼、超重及肥胖、红色及加工处理的肉类消费和过度饮酒。结直肠癌的发生和发展是一个历时数十年，多阶段进展，多因素参与，伴随一系列的分子事件的复杂进程。多年的研究揭示了大量的发生于DNA和RNA水平的分子特征性改变，形成了人们现在对结直肠癌发生的分子机制的认识,包括了基因组DNA序列的突变，DNA拷贝数的改变，启动子超甲基化，miRNA表达水平改变等，最终这些变化影响了结直肠癌蛋白质表达谱的改变。
影响结直肠癌预后的最重要因素就是早期诊断与早期治疗，研究显示TNM I期患者的五年生存率可达90%以上，而有远处器官转移的IV期患者5年生存率只有大约10%。虽然经过多年的基础和临床共同努力，结直肠癌死亡率和生存周期并未获得明显改善和延长，究其原因，在于相当一部分患者在就诊时就已经处于肿瘤进展期。有报道表明，30-40%的就诊患者已经存在区域侵润和转移性疾病，并无法通过单纯的手术治疗获得治愈，并且超过一半的患者术后复发并且罹难于肿瘤复发和转移。研究表明，通过对广泛人群进行筛查的结直肠癌早期检测和诊断，以及对早期结直肠癌进行干预，可以有效的延长生存期，而这一切依赖于高效而准确的结直肠癌诊断和预后生物标记物的发现及应用。
蛋白质参与了人类生理、病理过程的绝大部分活动，在疾病发生、机体反应和康复的各个进程都发挥着重要的作用，因此，作为生命活动的重要作用分子形式，蛋白质标志物自然是生物标志物筛选与鉴定的焦点。
iTRAQ技术是由美国ABI应用生物系统公司（Applied BiosystemsIncorporation, ABI)开发的同位素标记相对和绝对定量技术(isobaric tags forrelative and absolute quantitation, iTRAQ)的简称，相对于传统的定量鉴定，具有无可比拟的优点：在同一实验中，最多可以对多达4到8种不同样本进行蛋白定量比较，可以对样本中的绝大部分蛋白质进行标记，比如可以对磷酸化蛋白、糖基化蛋白等翻译后修饰蛋白进行定量、定性研究，从中获得更为详尽的样品信息，标记过程更简便，在室温条件下1小时即可完成，质谱检测更为灵敏。
本课题研究采用的蛋白组学研究策略是以同位素相对绝对定量技术（iTRAQ）为基础，通过联合多维固/液相色谱技术与串联质谱技术分离和鉴定蛋白质，对比结直肠癌与配对的正常对照粘膜组织蛋白质组定量结果和同一患者术前与术后血清蛋白质组定量结果，分别描绘结直肠癌患者组织对比和血清对比差异蛋白质谱，通过比对组织和血清的差异蛋白质谱，挑选一组在组织和血清中均有一致性差异表达的差异蛋白，经生物信息学分析后行Wester blot、免疫组化验证，筛选出一组具有理想诊断和/或预后价值或可作为治疗靶点的免疫组织化学标志物，以期提高结直肠癌疾病的早期诊断水平、改善患者预后和/或为治疗提供可能的靶点。相比于以往的研究，本课题的创新性体现在在一次iTRAQ标记实验中同时比对同一患者的组织与血清差异表达蛋白谱，筛选在组织学和血清学水平具有一致性差异的蛋白标志物，增强了后期验证结果的可靠性。
方法
本研究收集1例结直肠癌患者癌组织和配对正常粘膜组织，以及同一患者的术前和术后血清，经蛋白提取和经胰酶消化为多肽后，分别以同位素相对与绝对定量试剂盒（iTRAQ）标记组织与血清蛋白肽段，将二者混合后经过强阳离子交换柱（SCX）和反相色谱柱（RPLC）的二维分离，之后联用MALDI质谱技术对分离后的样品蛋白质多肽质谱图进行定量鉴定，经Mascot数据库检索，描绘结直肠癌患者组织与血清差异蛋白质谱。挑选出感兴趣的目的蛋白后，登录GeneOntcology与Swiss-Prot数据库进行差异蛋白质的亚细胞结构定位和功能分析。
经生物信息学分析，挑选出差异表达蛋白标志物，经Western blot和免疫组织化学法验证其表达水平。收集除用于iTRAQ定量的结直肠癌病例以外的3例与之临床病理特征匹配的结直肠癌病例，共4例的结直肠癌肿瘤组织及配对正常组织，经前述冰冻组织保存及蛋白提取方法，进行蛋白提取、定量后，分别取总蛋白各20μg进行Western blot验证，以PD QUEST软件半定量分析。收集80例结直肠癌患者肿瘤组织和匹配的正常对照粘膜组织，经福尔马林固定石蜡包埋处理后切片，经逐步脱蜡至水、双氧水（H2O2）封闭内源酶活性、高温高压抗原修复、10%牛血清封闭、抗原特异性一抗过夜孵育、漂洗后种属特异二抗常温孵育、DAB显色及苏木素复染后，由两位病理医师分别对免疫组化（IHC）结果进行双盲评分。IHC评分标准：在5-10个随机视野观察200个上皮细胞，从免疫阳性染色细胞比例和免疫染色强度两个方面进行综合评分，两位医师的评分平均后作为最终结果。根据免疫阳性细胞比例不同，分为：0分，阴性；1分，<10%阳性；2分，11%-50%阳性；3分，≥51%阳性。根据免疫染色强度不同，分为：0分，阴性；1分，弱阳性；2分，阳性；3分，强阳性。为系统评价蛋白标志物Prenylcysteine oxidase1和Peroxiredoxin-4的诊断和/或预后价值。为评价筛选的蛋白标志物的诊断可靠性，以免疫组化评分为依据，从低到高设立7个诊断阈值，分别是评分≥0、1、2、3、4、5和6。统计分析在不同的诊断阈值下，蛋白标志物用于结直肠癌（CRC）诊断的特异度、灵敏度、阴性预测值与阳性预测值、约登指数（Youden Index）和一致性检验Kappa值，绘制受试者工作特征曲线（ROC），确定界值点，计算ROC曲线下面积AUC。
结果
以同位素相对与绝对定量（iTRAQ）为基础、固/液相二维分离及MALDI质谱技术描绘结直肠癌组织与血清差异蛋白谱，经Scaffold筛选，NCBI_human库检索，共鉴定到血清蛋白198个,其中差异蛋白102个；肿瘤组织鉴定到蛋白568个，其中差异蛋白391个。经过比对发现，共有13个蛋白在血清和组织中都鉴定为差异表达蛋白，其中5个蛋白在肿瘤组织中表达水平上调，8个在肿瘤组织中表达水平下调，10个蛋白在术前血清中表达水平上调，3个蛋白在术后2周血清表达水平上调,4个蛋白表达差异具有一致性（我们规定，在肿瘤组织表达升高/降低和在术前血清表达升高/降低，称为表达差异具有一致性）。通过检索UniProt knowledgebase数据库(Swiss-Prot/TrEMBL, www.expasy.org)和GeneOntology (GO) Database数据库(http://www.geneontology.org/)，我们对筛选出的13个差异蛋白质的生物学功能进行了GO分析。GO分析结果显示此13个差异蛋白质，大部分（84.6﹪）具有两个或两个以上的分子功能或参与的生物学进程，其中与结合（Binding）功能有关的占84.6﹪，此外，还与生物功能调节（76.9%），细胞进程（69.2%），代谢进程（69.2%）和应激（84.6%）功能相关。我们选择了Prenylcysteine oxidase1， Peroxiredoxin-4，Lumican和Complement factor B这四个具有一致性差异表达的蛋白标志物进行Western-Blot实验，结果证实Prenylcysteine oxidase1， Peroxiredoxin-4这2个蛋白的表达量在肿瘤组织中高于正常组织。随后对这2个蛋白进行免疫组化验证，利用SPSS18.0软（卡方检验与Wilcoxon signed ranks检验）对免疫组化评分进行统计学分析。结果表明：与配对的正常对照组织相比，Prenylcysteine oxidase1和Peroxiredoxin-4在肿瘤组织的上调表达具有统计学意义（二者均P<0.01），二者分别在85%和71.25%的结直肠癌组织上调表达，卡方检验提示Peroxiredoxin-4的上调表达与淋巴结侵袭和TNM分期相关（P<0.05），Prenylcysteine oxidase1的上调表达与病例临床特征如年龄、性别、肿瘤部位、分化程度、肿瘤TNM分期和淋巴结侵袭之间并无显著相关性。经Logistic回归分析病例临床病理特征和蛋白标志物在肿瘤组织和正常组织的差异表达程度相关性，提示Prenylcysteine oxidase1和Peroxiredoxin-4在肿瘤组织和正常组织的差异表达程度均与淋巴结侵袭相关，OR值分别为3.386（95%可信区间，1.474-8.243）和2.858（95%可信区间，1.219-6.701）。Prenylcysteine oxidase1主要表达在胞浆，其阳性表达在95%的结直肠癌组织和60%的正常对照组织中被检测到。Peroxiredoxin-4的表达分布于胞浆和部分细胞外间质区域，其阳性表达可以在96.25%的结直肠癌组织和80%的对照正常组织样本中检测到。
Prenylcysteine oxidase1诊断结直肠癌（CRC）的诊断阈值是免疫组化评分≥3，经统计分析其CRC诊断特异度为93.75℅，敏感度为76.25℅，阳性预测值为92.4℅，阴性预测值为79.8℅，在该诊断阈值下，计算约登指数（YI）为70，收受者特征曲线（ROC）的曲线下面积（AUC）为89.4%。Peroxiredoxin-4诊断结直肠癌（CRC）的诊断阈值是免疫组化评分≥4，经统计分析其CRC诊断特异度为96.3℅，敏感度为63.8℅，阳性预测值为94.4℅，阴性预测值为72.6℅，在该诊断阈值下，约登指数为60.1，收受者特征曲线（ROC）的曲线下面积（AUC）为84.2%。
结论
我们初步描绘了结直肠癌组织与血清差异表达蛋白质谱，筛选出了一组具有较好诊断价值的蛋白标志物，验证了Prenylcysteine oxidase1和Peroxiredoxin-4在癌组织中的上调表达，为结直肠癌发生发展机制及肿瘤标志物的研究提供了新的线索。其中Prenylcysteine oxidase1对结直肠癌患者早期诊断以及Peroxiredoxin-4对结直肠癌转移相关的预后分析具有较大的价值。
Background and Aims
Colorectal cancer(CRC) is the third most commonly diagnosed cancer in males andthe second in females, with over1.2million new reported cases and608,700deathcases estimated to have occurred in2008according to WHO report. Incidence rateswere unbalanced distributed worldwide.The highest incidence rates are found inAustralia and New Zealand, Europe, and North America,whereas the lowest rates arefound in Africa and South-Central Asia. Rates are substantially higher in males thanin females.
In China the incidence and mortality of CRC is rapidly increasing in recent years,especially in big cities,mainly due to the raising of people’s living standards andchanging of people’s diets.Datas showed CRC is the third most commonly diagnosedcancer in the whole population of over1.3billion, the fourth in males and the third infemales, with over0.39million new cancer cases and0.19million deaths estimated tohave occurred in2009.
Several researches showed modifiable risk factors for colorectal cancer includesmoking, physical inactivity, overweight and obesity, red and processed meatconsumption, and excessive alcohol consumption. CRC develops through a complexprogressions of genetic and epigenetic abnormalities that transform normal epitheliuminto dysplastic epithelium, which may ultimately progress into invasive and metastaticdisease. Extensive molecular characterization of colon tumors at the DNA and RNAlevel has shaped our current knowledge about the molecular mechanisms thatinvolved in tumorigenesis,including DNA sequence mutations, DNA copy numberchanges, promoter hypermethylation, altered miRNA expression and so on, whichultimately affecting the spectrum of proteins expressed in CRC.
Despite of the advances in surgery-based comprehensive treatments for CRC, theoverall outcomes and survival terms of patients have not significantly improved or prolonged over the last few decades sinace most of the patients are diagnosed atadvanced stages.Therefore,diagnosis at early stage is considered as a crucial andpromising approach to reduce mortality and improve the prognosis.Reports haveshowed that the prognosis of CRC patients range dramaticly with five year survivalrates of more than90%for localized CRC (stage I) and only about10%for CRC thatmetastasized to distant organs (stage IV).Although considerable attentions and effortshave been spend on CRC, the reduced incidences of CRC and/or prolonged survivalterms of CRC patients were not gained during recent years, mostly due to the fact thatmost CRC patients were diagnosed at advanced stages. In fact,30–40%of patientshave regionally advanced or metastatic disease on presentation, which cannot becured by surgery alone. In addition, more than half of the patients develop recurrenceand die of the disease.It’s widely recommended to screen CRC in population as wellas interference at early stages,by which the survival terms of CRC patients could beprolonged.All these screening and early stages interference attempts are based onidentification of protein biomarkers with superior diagnosis effects.
Although biomarkers can adopt many molecular forms, proteins are major players indisease, response and recovery. Therefore, many efforts have been made to identifyprotein biomarkers.
ITRAQ is short for isobaric tags for relative and absolute quantitation,developed byApplied Biosystems Incorporation of USA. The iTRAQ is based on differentialisotopic labeling of proteins or peptides which are derived from two cell states (e.g.,healthy and tumor cells) with either light or heavy tags. This technique could label4components to the most in identification experiments simultaneously and had shownhuge advantages in throughput,sensitivity and quantitative precision to overcomesome drawbacks of traditional proteomic techniques and represented a powerfulalternative.
In the present work, iTRAQ was combined with two-dimensional liquidchromatography (2D-LC) and MS (Qstar-ESI, Applied Biosystems) to identifypotential biomarkers for CRC by two steps of comparison of protein profiles.First,weseperately compared protein profiles of CRC tissues and paired normal mucosal aswell as those of pre-surgical serum and post-surgical serum.Then the proteinsdifferentially quantificated in both tissue and serum sections were sorted out forfurther verification. The disregulated proteins were verified further by western-blot and immunohistochemistry（IHC）. The present study not only evaluated thesensitivity and specificity of the new potential biomarkers but also analysis thecorrelation and regression of the patients' clinical characteristic and the new potentialbiomarkers.Our work compared the disregulated proteins of CRC tissues and serumsto sort the protein biomarkers with accordant differential expression,this stragetycontributes to the reliablity of our results.
Methods
Tissue and serum specimens for iTRAQ were obtained from the same patient whounderwent colon cancer surgery in our hospital. CRC tissue and paired normalmucosal tissue(located at least5cm away from the tumor margin) were obtainedduring surgical resection. After excision, sample tissues were flash frozen at80°Cand stored until use.Serum samples were obtained1day before surgery and twoweeks after surgery. Peripheral venous blood samples(10ml) were taken byvenipuncture and allowed to clot.Then the serums were collected and stored at-80℃until use.
The iTRAQ labeled samples were fractionated by a SCX column and RP-LC followedby ESI-MS/MS analysis.Protein identification and quantification for the iTRAQexperiment were performed with Mascot version2.3.01(Matrix Science Inc,USA).The Scaffold version3.0(Proteome Software Inc,Portland) in Mascot software wasused for peptide identification and isoform specific quantification.
The sub-cellular location and function of the identified proteins were elucidated bythe UniProt knowledgebase (Swiss-Prot/TrEMBL,www.expasy.org) and the GeneOntology(GO) database(http://www.geneontology.org/).CRC tissue and paired normalmucosal tissue samples used in iTRAQ exprement and other3cases of CRC tissuesand paired normal mucosal tissues containing20μg of total proteins were tested bywestern blot.Two proteins from the biomarker panel analysis were selected for IHCverification using an independent and larger sample set(80cases).Immunopositivestaining was evaluated in five areas.Sections were scored as positive if epithelial cellsshowed immunopositivity in the cytoplasm, plasma membrane, and/or nucleus whenjudged independently by two scorers who were blinded to the clinical outcome.Aquantitative score was performed by estimating the percentage of immuno-positivestained cells:0, negative;1,<10%positive cells;2,11%to50%positive cells;3,≥51%positive cells. Meanwhile, the intensity of staining was scored by evaluating the average staining intensity of the positive cells (0, negative;1, weak;2, moderate; and3, strong). Finally, a total score(ranging from0to6) was obtained by adding thequantitative score and the intensity score for each of the160sections.Statisticalanalyses were carried out with SPSS19.0software (SPSS, Chicago, IL, USA).Wilcoxon signed-rank test was used to compare qualitative variables of IHC resultsbetween CRC tissues and paired normal mucosal tissues. Pearson χ2or correctionalχ2tests were used to analyze the correlation between the differential expression ofindicated proteins and patients’ clinicopathologic characteristics.Logistic regressionwas used to analysis risk factors that could effect expressions of potential proteinbiomarkers. Receiver operating characteristic (ROC) curves were used to determinethe diagnostic values of the markers.
Results
iTRAQ agents were used in the present study to label digested peptides for comparingprotein expressions between tissues of CRC and matched normal mucosal as well asthat between serums of pre-and post-surgical. Here, up-or down-regulation is statedas protein expression in CRC tissues relative to paired normal mucosa or inpre-surgical serums relative to post-surgical serums.A total of568proteins(391differentially expressed) were identified in tissues and198proteins(102differentiallyexpressed) in serums. Using the cut-off value of1.5-fold for up-regulation anddown-regulation, a total of187proteins(73up-regulated and114down-regulated) and44proteins(19up-regulated and25down-regulated) were identified as differentiallyexpressed in tissues and serums,seperately.13proteins differentially expressed in bothtissue and serum sections were sorted out for further analysis.
UniProt knowledge base (Swiss-Prot/TrEMBL) and the Gene Ontology (GO)database annotated that10of the13differential proteins were located in more thanone cellular component or organelles. In brief,38.5%,7.7%,69.2%,69.2%,23.1%,53.8%and53.8%of the13differential proteins were located inthe organelle, organelle part,extracellular region,extracellular regionpart,macromolecular complex, cell and cell part,respectively. We analyzed the proteinfunction annotated by the UniProt knowledgebase (Swiss-Prot/TrEMBL) and the GOdatabase.The following biological processes and molecular functions were involvedmore frequently:binding (84.6%), biological regulation(76.9%), cellular process(69.2%), metabolic process(69.2%), and response to stimulus (84.6%). In total,84.6% differential proteins were involved in more than one molecular function andbiological process.
To validate our proteomic results, we selected4proteins, with the accordantdifferential expression of tissues and serums,out of13differentially expressedproteins and directly assessed their levels of expression by westernblot.Prenylcysteine oxidase1and peroxiredoxin-4were validated to be up-regulatedin4CRC cases.Immunohistochemical verifications of prenylcysteine oxidase1andperoxiredoxin-4were tested in80cases of primary human CRC tissues and theirpaired normal mucosa.The scores of prenylcysteine oxidase1and peroxiredoxin-4between CRC tissues and normal mucosa show statistically significant differences.(Wilcoxon signed-ranks test, P<0.001for prenylcysteine oxidase1, P<0.001forperoxiredoxin-4). Compared with normal cells, prenylcysteine oxidase1andperoxiredoxin-4were verified to be up-regulated in85%and71.25%of the CRCcases, respectively. As the results of Pearson χ2or correctional χ2tests, theup-regulated expression of peroxiredoxin-4was found to be correlated with lymphnodes invasions and TNM stages.Interestingly, logistic correlation tests showed thatdifferential expression of both prenylcysteine oxidase1and peroxiredoxin-4betweenCRCs and normal mucosa samples were correlated with lymph nodes invasions withOR of3.386(95%Wald Confidence Limits,1.474-8.243) and2.858(95%WaldConfidence Limits,1.219-6.701),respectively.Prenylcysteine oxidase1expressionshowed cytoplasm expression,which was detected in95%of CRC samples and60%of paired normal mucosa samples.Peroxiredoxin-4showed cytoplasm andextracellular expression,which was detected in96.25%of the CRC samples and80%of matched normal mucosa tissues.Cut-off values for prenylcysteine oxidase1andperoxiredoxin-4were stastically analyzed and determined as score3and4,respectively, to distinguish CRC from paired normal mucosa by comparing the sumsof sensitivity and specificity of various diagnosis criterias; these criterias were set as I,II, III, IV, V, VI and VII (scores≥0,1,2,3,4,5and6,respectively). Relateddiagnostic values for CRC detection using prenylcysteine oxidase1andperoxiredoxin-4were shown in Table4. The areas under the curve (AUC) of thereceiver operator characteristic (ROC) curves of prenylcysteine oxidase1andperoxiredoxin-4were89.4and84.2, respectively. These results showed moderatediagnostic accuracies for prenylcysteine oxidase1and peroxiredoxin-4.
Conclusions
We performed the iTRAQ-based CRC proteomic research and identified a total of391differentially expressed proteins in tissues and102differentially expressedproteins in serums. Prenylcysteine oxidase1and peroxiredoxin-4were validated to beup-regulated in CRC compared with paired normal mucosa by western blot andimmunohistochemistry. Our results suggest that prenylcysteine oxidase1andperoxiredoxin-4are potential diagnostic or prognosis biomarkers for CRCs, and theymay play an important role in tumorigenesis and development of CRCs.
引文
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