摘要
膀胱癌是泌尿系统威胁人类健康最常见的恶性肿瘤之一,其发病率及死亡率不断上升。世界范围内,膀胱癌位列男性最常见实体瘤的第四位,在女性位列第七位,每年新诊断的膀胱癌患者超过350,000名。美国癌症协会统计2010年美国膀胱癌新发病例为70,530例,死亡病例为14,680例。在我国,膀胱癌目前仍是最常见的泌尿系统恶性肿瘤,且发病率呈现稳中有升的趋势。临床特点为一旦肿瘤侵袭浆膜层则复发率高、预后差。膀胱癌患者中超过90%为尿路上皮癌,首次确诊为膀胱癌的患者中,55%-60%为低级别尿路上皮癌。低级别尿路上皮癌即使经过适当的腔内或开放治疗,仍有很高的复发率。通常,复发仍为分化较好的低级别尿路上皮癌,但仍有16%-25%的患者肿瘤病理分级增加,且大约10%低级别尿路上皮癌可发展为肌层浸润或远处转移。膀胱低级别尿路上皮癌发生发展是一个多因素、多基因及多途径改变的复杂过程。
在目前临床工作中,膀胱镜检查是发现,诊断及监测膀胱癌复发和进展的金标准。反复的膀胱镜检查在疾病早期阶段促使患者接受有效的治疗,因此潜在地减缓了疾病向肌层侵袭的进展。然而,膀胱镜是一种侵袭性、费时和昂贵的检查手段,不能够很好地被患者接受和配合。尿脱落细胞学检查仍是辅助膀胱镜的一种非侵袭性的检查手段,特别是在发现高侵袭性膀胱癌方面具有很高的敏感度,但在低度恶性的病变方面其敏感度较低,并且它的准确性依赖于病理医师的经验。因此,在筛查、早期发现和预测浅表肿瘤向侵袭性进展方面,致力于寻找具有高敏感度和特异度、可信赖的非侵袭性标记物具有重要的临床意义。迄今为止,临床常规流程中,仍没有敏感度和特异度高的血清或尿液肿瘤诊断标志物用以发现及跟踪随访膀胱癌患者。
虽然基于基因水平的基础研究可以为膀胱癌疾病的发生,发展分子机制的解读提供有力的依据,但是这些研究往往强调单个或者少数几个分子的作用,缺乏蛋白质水平的整体性、整合性及系统性的研究。归根结底,蛋白质是生命活动的直接执行者,且某些基因的表达产物可能不只一个,而且基因的表达方式错综复杂,同样的一个基因在不同的机体周围环境和不同的机体生理状态下,会产生不同的蛋白质,发挥完全不同的作用。基因结构和功能的稳定性与蛋白质的复杂性和多变性存在巨大的反差。此外,对于功能蛋白质,还涉及到蛋白质的后期加工、修饰以及转运定位的过程,这些过程是基因不能决定的；但这些过程中的任何一个步骤发生微细的差错即可导致机体的疾病。另外,miRNA的发现也使我们意识到即使是在mRNA上表达有差异,但由于miRNA对转录后的基因表达调控,也有可能在相应的蛋白质水平上表达有差异。因此,膀胱癌疾病蛋白水平的研究是不容忽视的重要课题。
蛋白质组是一个在时间和空间上动态变化着的整体,其目的是从整体的角度分析细胞内动态变化的蛋白组成成分,表达水平与修饰状态,以及蛋白质之间的相互作用与联系,揭示蛋白质功能与细胞生命活动规律。研究体液的蛋白质组学模式的出现在早期发现癌症方面寻找新的、高敏感度的诊断工具提供新的机会。临床蛋白质组学领域的主要目的是在体液中寻找能够鉴别疾病的标志物,可以相对廉价的检测并早期诊断疾病。目前而言,血清或血浆蛋白质组学受到广泛关注。由于尿液可直接接触肿瘤并且样本易于获取,在疾病的早期发现及疾病监测方面,尿液相对于用血浆来发现膀胱癌潜在的标志物而言是一个有吸引力的选择。尽管现在一些同时存在于血液和尿液中的蛋白已经被作为膀胱癌的标记物,如膀胱癌抗原、核基质蛋白和纤维蛋白原代谢产物；但是其特异性和敏感性都欠理想。运用蛋白组学技术从尿液样本中来研究膀胱癌尿液相关蛋白以筛选,诊断及监测预后是目前研究重要的趋势。
目前为止,已有不少研究运用双向凝胶电泳和质谱鉴定技术以识别在膀胱癌尿液中差异表达的蛋白,并且应用考马斯染色、银染或放射标记染色法标记尿蛋白提取物。如2009年Feldman. et al运用蛋白质组学策略分析了膀胱癌患者和正常对照人群的尿液差异蛋白,并对兴趣蛋白Cystatin B进一步在组织中应用免疫组织化学技术和在尿液中用半定量的Western blot技术进行分析。研究结果发现尿液中Cystatin B的表达水平与肿瘤的分级(P=0.062)，分期(P=0.0047)及肿瘤复发(P=0.0104)和进展(P=0.0007)的时间正相关。2011年Li. et al通过运用双向电泳筛选健康志愿者和低度恶性及侵袭性膀胱癌患者的尿液样本,以期识别能够早期检测膀胱癌的生物标记物。他们观察到纤维蛋白原、乳酸脱氢酶B、载脂蛋白A1、丛生蛋白和触珠蛋白等5个蛋白在低度恶性或侵袭性膀胱癌患者尿液中表达增高。进一步对尿液样本分析后发现,相对于低度恶性膀胱癌,载脂蛋白A1在侵袭性膀胱癌中表达显著增高。应用ELISA法测定载脂蛋白A1的水平,发现在18.22ng/ml水平其具有可以从正常人群中辨别膀胱癌患者的诊断效能(敏感度和特异度分别为91.6%和85.7%),并认为其浓度在29.86ng/ml水平可辨别侵袭性膀胱癌患者和低度恶性膀胱癌患者(敏感度和特异度分别为83.7%和89.7%)。
然而,随着蛋白质组学技术的改进,荧光差异双向凝胶电泳技术为基础的蛋白质组学研究是研究的趋势。荧光差异凝胶电泳技术可在同一块胶上同时分离多个分别由不同荧光标记的样品。由于不同的荧光标记样品有不同的激发波长,可通过不同的滤光片记录互不干扰的胶图结果。由于有了多色荧光标记,使得在同一块胶中分离并分析多个样本成为可能。这样有效避免了不同胶间的系统误差,特别适合比较不同样本间差异。1997年,Unlu等发表了第一篇荧光差异凝胶电泳的论文,将不同的样品分别用不同的荧光染料进行标记后混合在同一块凝胶中进行双向电泳,极大地提高了实验结果的重复性和定量的准确性。据我们所知,应用荧光标记尿蛋白提取物进行双向凝胶电泳的技术报道比较罕见。仅Orenes-Pinero等通过研究发现可以运用荧光差异双向凝胶电泳蛋白组学策略来探索诊断膀胱癌的尿液标记物。
本研究正式立足于尿液蛋白质组学水平,建立规范和标准的收集临床标本工作流程,制定科学合理的纳入和排除标准,在收集足够膀胱癌患者和健康对照人群尿液样本的基础上,按照病理诊断结果进行分组(低级别尿路上皮癌组,高级别尿路上皮癌组和浸润性尿路上皮癌组),利用反复超滤和除盐份的方法浓缩,纯化尿液蛋白,利用荧光差异双向凝胶电泳进行筛选及分离,电泳后用Typhoon9400成像系统扫描凝胶并用DeCyder软件分析差异蛋白。运用基质辅助激光解吸电离飞行时间技术对这些差异蛋白进行鉴定,联合生物信息学方法以选取感兴趣的候选蛋白(Gc-globulin)。待对候选蛋白验证其结果可靠性后,对大量临床膀胱癌患者尿液和对照组尿液样本进行ELISA定量检测GC的浓度,结合已检测临床标本的临床资料进行统计分析以建立候选蛋白分子标记物GC与膀胱癌疾病的联系,进一步确定有效诊断和监测膀胱癌疾病的蛋白分子标记物,对于研究膀胱癌的发病机制,诊断方法,疾病监测和治疗方法等方面的研究奠定基础。
目前运用荧光差异双向凝胶电泳的蛋白组学技术研究膀胱癌尿液蛋白的文献报道比较少,为此开展筛选膀胱癌尿液新标记物Gc-globulin的荧光差异蛋白质组学研究正是立足于从尿液蛋白水平获取理想的膀胱癌诊断,进展及预后分子标记物,阐述相关的发病机制。无疑对深入了解膀胱癌疾病的发生,发展及预后问题,是具有重要的理论及现实意义的研究项目。
研究方法
(一)临床资料和标本收集：
(1)膀胱癌组尿液标本来自南方医科大学南方医院泌尿外科住院患者。所有膀胱癌的患者经术前CT或逆行肾盂输尿管造影来确定上尿路没有病变。这些患者都有最初诊断的阳性结果或者膀胱镜检查指证。膀胱镜检查前收集患者尿标本。正常对照组尿液标本来南方医科大学南方医院体检中心健康人群。尿液标本收集后立即冷却到4℃,然后在4个小时内转移到-80。C储存。详细登记好病人的临床资料特别是膀胱镜检查组织病理结果,术后病理结果及临床诊断结果。患者自愿捐献并签定知情同意书。本研究经南方医院伦理道德委员会批准。排除标准：1：伴有肾脏疾病尿蛋白阳性的患者；2：伴有除膀胱癌外其他泌尿系肿瘤的患者；3：伴有不明原因的肉眼血尿的患者(电泳时)；4：伴有严重的泌尿系感染的患者(电泳时)。
(二)候选蛋白标记物的筛选
(1)选取正常对照组和膀胱癌组尿液标本各12例,其中低级别尿路上皮癌,高级别尿路上皮癌和浸润性尿路上皮癌样本各4例。将样本从-80℃冰箱拿出,融解后即刻添加蛋白酶抑制剂(50：1),采用反复超滤离心洗涤的方法联合除盐方法浓缩,纯化尿液蛋白。用Bradford方法进行蛋白浓度测定。
(2)运用双向凝胶电泳构建稳定性好,重复性高的人尿液双向电泳图谱：
(2.1)蛋白样品和上样缓冲液共450μL充分混合,用24cm IPG干胶条采取被动泡涨13h,等电聚焦条件为500V1h,1,000V1h,5,000V1h,8,000V60KVh,500V3h。聚焦完毕后将胶条先后平衡两次,随后将胶条移至12.0%的SDS-PAGE胶上端进行第二向电泳,直至溴酚蓝达胶底线。
(2.2)银染40.0%乙醇,10.0%冰醋酸固定2次,每次15min；30.0%乙醇,0.2%Na2S2O3,6.8%乙酸钠敏化30min；蒸馏水洗3次,每次5min；2.50%oAgN03染色20min；蒸馏水洗2次,每次1min;2.5%Na2CO3,0.04%o甲醛显影至蛋白质点清晰；立即加入5.0%冰醋酸终止10min；蒸馏水洗3次,每次5min；最后用30.0%乙醇,4.6%甘油保存。
(2.3)凝胶通过UMAX PowerLook1100投射扫描仪进行扫描获取2-DE凝胶图像,利用Melanie4分析软件对图像进行分析。
(3)运用荧光差异双向凝胶电泳技术对正常对照组和膀胱癌组尿液蛋白进行分离及筛选差异蛋白：
(3.1)将正常对照组和膀胱癌组尿液组各组样品pH值调至8.5；每份样本50μg(5～10μl)避光条件下加入1μl (400pmol)的CyDye Cy3、Cy5、Cy2荧光染料工作液,其中Cy2作为内标标记两组尿液蛋白样品混合物,Cy3标记健康对照组尿液蛋白样品,Cy5标记膀胱癌组尿液蛋白样品。
(3.2)将标记荧光染料的样品进行双向电泳,步骤与普通双向电泳相同。
(3.3)直到溴酚蓝到达胶的底端结束双向电泳。用Typhoon9400成像系统扫描荧光凝胶,各荧光染料的激发光和发射光波长分别为：Cy2:480nm和530nm, Cy3:540nm和590nm, Cy5:620nn和680nm。扫描后的图像文件用DeCyder5.0版本软件分析,应用BVA模式进行胶与胶之间的匹配,不同组间选择Student-t检验。挑选出两组间具有统计学意义且差异倍数>2.0的显著增加或者显著减少的感兴趣差异蛋白点。
(3.4)取对照组及实验组蛋白样品各500μg,按常规方法进行制备胶双向电泳后,经考马斯亮蓝染色,用机械手臂挖取与感兴趣差异蛋白相匹配的蛋白点。对所挖取蛋白点用50%乙腈冲洗、胰蛋白酶消化、并在靶条上进行标肽和蛋白样品点样后,应用基质辅助激光解析／电离飞行时间质谱分析蛋白质肽质量指纹谱,并将结果输入NCBI和SWISS-PROT蛋白质数据库进行检索。
(三)应用生物信息学方法选定候选蛋白GC
借助生物信息学研究手段如：SWISS-PROT, String和Pubgene等工具对所鉴定蛋白分析所参与的生物学过程、蛋白质间的相互作用及蛋白质特有的结构和功能等,以便选取候选蛋白进行进一步研究。
(四)候选蛋白GC在健康人群和膀胱癌患者尿液中的表达
(1)候选蛋白的Western Blot验证：选取正常对照组尿液标本8例；膀胱癌组尿液标本8例,其中低级别尿路上皮癌2例,高级别尿路上皮癌2例和浸润性尿路上皮癌4例。每个样本各自提取尿液总蛋白定量后,每孔加入约30μg蛋白,10%SDS-PAGE凝胶电泳,蛋白转移到PVDF膜上,5%脱脂奶粉的TBS-T室温封闭1h,封闭一抗、二抗后由ECL化学发光试剂盒检测,压底片曝光后显影。
(2)选取144例尿液样本运用ELISA方法检测GC浓度,其中正常对照组42例；膀胱良性病变11例和膀胱癌组91例。根据世界卫生组织膀胱癌的分类标准：浸润性尿路上皮癌23例和非浸润性尿路上皮癌68例(低级别尿路上皮癌38例,高级别尿路上皮癌30例)。
(五)候选蛋白GC浓度与相关临床资料的统计分析
待健康人群和膀胱癌患者中候选蛋白浓度定量检测后,根据临床资料如：年龄,性别,肿瘤的原发/复发,转移/未转移,伴/不伴血尿,病理分级等进行亚组分析；还有通过ROC曲线来确定用于诊断膀胱癌/区分浸润性膀胱癌的cut-off值,并计算相应的特异度和敏感度。
(六)候选蛋白GC在正常对照和膀胱癌组织中的表达
(1)差异表达蛋白的Western Blot验证：对单个的正常对照组和膀胱癌组织样本运用液氮研磨的方法提取总蛋白进行免疫印迹检测。
(2)差异表达蛋白的免疫组织化学技术验证：选取正常对照组标本10例；膀胱癌标本26例的石蜡包块进行研究。其中低级别尿路上皮癌8例,高级别尿路上皮癌8例和浸润性尿路上皮癌10例。石蜡切片常规脱腊入水,接着用3%H202—甲醇10分钟后,水洗,再用PBS洗3次后,加生物素标记的特异性一抗,37℃30分钟,置湿盒内。PBS洗3次,加酶标记的卵白素,37℃30分钟,PBS洗3次,DAB显色后,流水冲洗,复染、脱水、封片。
研究结果
1.对收集的人尿液样品进行反复超滤及除盐优化处理后,可成功地获得人尿液样本分辨率高、重复性好的双向电泳图谱；
2.成功构建了膀胱癌组和正常对照组尿液样本的2D-DIGE图谱,对其中24个差异超过2倍的差异点进行质谱鉴定,共鉴定出16个不同的蛋白。其中ALB, GC, HP, FGB, APOA1, RBP4和SECTM1等7个蛋白在膀胱癌组中的尿液样品中高表达,剩余UMOD, KNG1, AMY1A, AMY2A, ITIH4, AMBP, HSPG2, CST5和MASP2等9个蛋白在膀胱癌组中低表达；
3. String和Pubgene等生物信息学分析提示GC蛋白在生长,发病机理,分泌,翻译,细胞凋亡,死亡,消化及信号转导等生物过程起着重要的作用,且GC蛋白与其他已鉴定的差异蛋白有着密切的联系,可作为候选蛋白；
4.GC蛋白的Western blotting结果比较发现GC在膀胱癌组尿液样品中的表达水平要高于正常对照组,有显著性差异。Western blotting结果与蛋白质组学的结果一致。
5.膀胱癌组织中的GC蛋白的表达要高于癌旁组织的表达。通过两组的免疫组织化学结果也发现：GC在移行上皮细胞和肿瘤细胞的胞浆中表达,染色较正常对照组强。提示GC在膀胱癌组中表达上调；
6.膀胱癌组GC蛋白浓度为1013.70±851.25ng/mg,良性病变组GC蛋白浓度为105.32±47.81ng/mg,正常对照组GC蛋白浓度为99.34±55.87ng/mg。膀胱组与良性病变组和正常对照组均有统计学差异(P<0.05),但良性病变组和正常对照组之间没有统计学差异(P>0.05)。按照病理级别分组,低级别尿路上皮癌GC蛋白浓度为472.92±348.02ng/mg,高级别尿路上皮癌GC蛋白浓度为1014.06±753.16ng/mg,浸润性尿路上皮癌GC蛋白浓度为1906.69±840.86ng/mg;且三组间均有统计学差异(P<0.05)。
7.ROC分析示采用161.086ng/mg GC蛋白浓度来筛选膀胱癌,相应的敏感度和特异度为92.31%和83.02%；当尿液中GC蛋白浓度大于1407.481ng/mg时,提示为浸润性膀胱癌,其敏感度和特异度为82.61%和88.24%。
结论
1.建立了稳定性高,重复性较好的人尿液蛋白组双向凝胶电泳技术和方法,为人膀胱癌尿液蛋白质组学进一步研究奠定了基础；
2.运用荧光差异蛋白组学方法比较了膀胱癌组和正常对照组尿液样本蛋白图谱；
3.对24个差异蛋白点进行质谱鉴定,共鉴定出16个不同的蛋白。其中ALB, GC, HP, FGB, APOA1, RBP4和SECTM1等7个蛋白在膀胱癌组中的尿液样品中高表达,剩余UMOD, KNG1, AMY1A, AMY2A, ITIH4, AMBP, HSPG2, CST5和MASP2等9个蛋白在膀胱癌组中低表达；
4.生物信息学分析提示GC蛋白可作为候选蛋白进一步研究；
5.GC蛋白在膀胱癌组织中的表达要高于癌旁组织的表达,胞浆表达；
6.GC蛋白在膀胱癌尿液中显著升高表达,且与膀胱癌病理分级正相关；
7.GC蛋白可以作为筛选和有效监测膀胱癌的一个潜在尿液蛋白标记物。
Bladder cancer is one of the most common malignant tumors of the urinary system threating to human health, and with its morbidity and mortality rising. All over the world, bladder cancer is the fourth most common cancer in men and the seventh most common among women, and there are more than350,000bladder cancer patients diagnosised each year. As is estimated by American Cancer Society statistics, In2010, around70,530new cases and14,680deaths from bladder cancer occurred in the United States. Bladder cancer is still the most common malignant tumors of the urinary system in China, and presents the trend of steadily morbidity. In general, it is clinically characterized by high recurrent rates and poor prognosis once tumors progress to muscularis propria invasive disease. At diagnosis,～90%of bladder cancers are urothelial cancer,55%to60%of them are low grade urothelial carcinoma. Some patients with the low grade urothelial carcinoma will recur even after appropriate treatment of the cavity or open. Typically, the patients recurred have a better differentiation of low grade urothelial carcinoma, but16%to25%of the patients will have the tumor grade increased. Furthermore, around10%of low grade urothelial carcinoma will invade deeper layers or distant transfer. On the whole, the development of bladder low gradel urothelial carcinoma progressing is a multifactor, multi-gene complex process and multi-channel change.
Currently, cystoscopy and bladder biopsy are considered as the most reliable methods for diagnosis and surveillance of bladder cancer. Repeated cystoscopy enable patients to receive effective treatment in the early stages of disease, potentially slowing disease progress myometrial invasion. However, cystoscopy is an invasive, time-consuming and expensive examination, which can not be accepted well for patients. Urine cytology, a non-invasive means, is still assisted with cystoscopy, especially in the highly invasive bladder cancer with a high sensitivity. But its sensitivity is lower in the low-grade lesions, and its accuracy is dependent on the experience of the pathologists. Improving the predictive ability would greatly benefit treatment of patients and monitoring of their condition. Thus, the development of a reliable non-invasive biomarker would be highly valuable for increasing the early detection rate of bladder cancer and predicting the progression of superficial tumors in time. To date, during the clinical routine process, we still do not have a good sensitivity and specificity of serum or urine tumor diagnostic markers for the detection and tracking bladder cancer patients.
The basic research based on the genetic level has provided a strong basis for the occurrence of bladder cancer diseases and the development of the interpretation of the molecular mechanisms, but these studies emphasized the role of a single or a few molecules, lacking of integrity of the protein level and systemthe nature of the study. Ultimately, the protein is a direct execution of the life activities, and the expression of certain genes in the product may be more than one. Moreover, the expression for gene is complicated. The same gene with different surrounding environment of the body and different physiological states of the body, will produce different the protein and play an entirely different role. There is a great contrast on gene structure with stability compared to the complexity and vari of the protein function. In addition, for the functions of proteins, it is also related to the post-processing of the protein, modification and transfer the process of positioning, which these processes can not be decided by the gene, once, any one of these process steps with any little error can result in the body's disease. At last, discovery of miRNA allows us to realize differentially expressed miRNA post-transcriptional regulation of gene expression, is also possible in the corresponding protein levels. Therefore, studying bladder cancer disease on the protein level is an important issue that should not be overlooked.
The Proteome that is a dynamic whole changing with time and space is aimed at analyzing the composition, expression levels and modification state, interaction and association of the proteins in cells from a holistic a holistic perspective, and revealing the functions of proteins as well as laws of cellular activity. The models of humoral proteomics research provide new opportunities to find new, highly sensitive diagnostic tools for the early detection of cancer. The main purpose of the clinical proteomics research is looking for a marker to identify disease in body fluids, which can be relatively inexpensive detection and early diagnosis of disease. Currently, serum or plasma proteomics have attracted widespread attention. While, the urine can directly contact with the tumor samples, so it is readily available in the early detection of disease and disease surveillance. Therefore, urine is an attractive option to bladder cancer finding potential markers compared to plasma. Although there are some known protein markers of bladder cancer identified in the blood and urine such as bladder cancer antigen, nuclear matrix proteins and fibrinogen metabolites; they were unsatisfactory specificity and sensitivity. To sum up, studies using proteomics technology for urine analysis diagnosis and monitori prognosis bladder cancer is a current research trend
To date, many studies use two-dimensional gel electrophoresis and mass spectrometry techniques to identify differentially expressed proteins in the urine of bladder cancer, using Maas staining, silver staining or radiolabeled staining marked urinary protein extracts. In2009, Feldman et al conducted a proteomics strategy analysis of the urine of bladder cancer patients and normal control subjects to find differently expressed proteins, and the protein of interest Cystatin B was further tested by immunohistochemical techniques in the organization and semi-quantitative Western blot techniquein urine usin. They found the expression level of urine Cystatin B is positively associated with tumor grade (P=0.062), stage (P=0.0047) and tumor recurrence (P=0.0104) and the progress of time (P=0.0007). In2011, Li et al performed a study by using two-dimensional electrophoresis screening healthy volunteers, low-grade and invasive bladder cancer urine samples in order to identify capable of early detection of bladder cancer biomarkers. They found that the fibrinogen, lactate dehydrogenase B, apolipoprotein Al, profusion protein and haptoglobin5protein expression increased in the urine of patients with bladder cancer of low malignant or aggressive. After further analysis of the urine samples, apolipoprotein A1expression was significantly higher in invasive bladder cancer compared to the low grade bladder cancer. Apo-A1level was measured quantitatively using ELISA and was suggested to provide diagnostic utility to distinguish patients with bladder cancer from controls at18.22ng/ml with a sensitivity and specificity of91.6%and85.7%respectively, and at29.86ng/ml for distinguish patients with low malignant bladder cancer from patients with aggressive bladder cancer with a sensitivity and specificity of83.7%and89.7%respectively.
However, with the improvement of proteomics technology, fluorescent differential two-dimensional gel electrophoresis-based proteomics research is a new trend. Two-dimensional fluorescent differential gel electrophoresis (2D-DIGE) is an advanced sensitive gel-based separation and quantification approach. Protein samples are prelabeled with different fluorescent dyes, mixed and run simultaneously on the same gel. Different excitation wavelengths can be recorded by different filters the Noninterference gum diagram results due to the different fluorescent labeled sample.
This method could effectively avoid systematic errors between different gels, especially suitable for the differences between the comparison of different samples. In1997, Unlu etc. firstly published papers about the fluorescence difference gel electrophoresis, which the different samples with different fluorescent dyes labeled and mixed in the same gel and then were conducted a two-dimensional electrophoresis, greatly improving the experimental results of repeated and quantitative accuracy. As far as we know, the study for bladder cancer by labeling urinary protein extracts and using the fluorescent two-dimensional gel electrophoresis is rare. Only in2007, Orenes-Pinero used2D-DIGE proteomics strategy to explore urine markers for diagnosis of bladder cancer.
In our present study, from the view of the formal level of urine proteomics, based on the establishment of norms and standards for the collection of clinical specimens workflow, developing the scientific and rational inclusion and exclusion criteria, then, we collected the enough the urine samples form patients with bladder cancer and healthy control subjects. The diagnosis was conducted by two different pathologists in Nanfang hospital of Southern Medical University according to the criteria of World Health Organization classification of tumour. The bladder cancer patients were divided into the low-grade urothelial carcinoma group, the high-level urothelial carcinoma group and the invasive urothelial carcinoma group. We used the method of repeated ultrafiltration and the salt removing to purify the total urine protein. Two-dimensional fluorescent differential gel electrophoresis was used for screening and separation. After electrophoresis, the gel was scaned by Typhoon9400imaging system and analyzed using DeCyder software to find the differently expressed proteins. The proteins were identified using matrix-assisted laser desorption ionization time-of-flight technology. Then, we applied bioinformatics methods to select the interested candidate proteins (GC). After the verification of the result of the candidate protein reliability, a large number of clinical bladder cancer urine of patients and the control group urine samples was used to measure GC protein concentration by ELISA. Statistical analysis was performed by ELISA results combined with the clinical data of clinical specimens to establish the relationship between the candidate protein molecular markers and bladder cancer disease so as to further determine the effective diagnosis and monitor of bladder cancer disease, study the pathogenesis of bladder cancer. These will be a basis for the study to diagnosis, disease monitor and treatment of bladder cancer.
However, up to date, studies using two-dimensional fluorescent differential gel electrophoresis for urine analysis related to bladder cancer are scarce. Therefore, to our present research, Identification of urinary Gc-globulin as a novel biomarker for bladder cancer by two-dimensional fluorescent differential gel electrophoresis (2D-DIGE) is to acquire the ideal biomarker for the early detection, development and prognosis of bladder cancer on the level of urine samples. Undoubtedly, this project has important theoretical and practical significance on the in-depth understanding of the occurrence, development and prognosis of bladder cancer.
Material and methods
1. Specimen collection and preparation
Studies were done with the approval of the bioethics committee of Nanfang hospital. All subjects were informed about the purpose of the study and gave their written consent. All patients had their upper tracts cleared via examinations, healthy volunteers with no evidence of disease were used as control group. All subjects were recruited from the Chinese Han population at Nanfang hospital from January2011to April2012. Pre-cystoscopy voided urine specimens were collected from patients presenting positive findings under suspicion of bladder cancer. Bladder cancer tissue and normal urothelial tissue were harvested form the cystectomy specimens of individual patients. The bladder cancer was confirmed by cystoscopy combined with histopathological information after subsequent surgical operations. The diagnosis was conducted by two pathologists in Nanfang hospital of Southern Medical University according to the criteria of World Health Organization classification of tumours. Each urine sample (20ml) was collected into a sterile plastic tube and then immediately centrifuged at1500x g for5min at4℃to remove cell debris and particulate matter. The supernatant was stored at-80℃for further analysis.
2. The screening of the candidate protein marker
(1) Equal volume urine specimens from12bladder cancer patients and12controls were pooled respectively for2D-DIGE analysis. Of these bladder cancer patients, there were8cases of non-invasive papillary urothelial carcinoma (4were low grade and4were high) and4cases of infiltrating urothelial carcinoma. For processing, samples were first thawed on ice, adding protease inhibitors,1mmol/L phenylmethylsulfonyl fluoride,5mmol/L phenanthroline, and5mmol/L benzamidine, and then centrifuged using Centricon Plus-20,10,000MWCO devices. Then proteins in the concentrated urine were precipitated using a ReadyPrep2-D clean up kit to remove other interfering components according to the manufacturer's instructions. Protein concentration was measured by using the Bradford method.
(2) To establish the good stability, high repeatability urine proteomic profiles by two-dimensional gel electrophoresis
(2.1) The Immobiline Dry strip (pH3-10, length24cm) was rehydrated with
100pg protein in450ml was rehydrated buffer for13h at room temperature. The first dimensional electrophoresis was performed on Protean IEF cell with a total of60kVh, The soelectric focusing conditions were500V1h,1,000V1h,5,000V3h,8,000V60KVh,500V3h.Then the strip was subjected to two-step equilibration for each step15min. The second dimensional electrophoresis was carried out in a homogeneous SDS-PAGE (12%) using a Protean II xi2D cell until the bromophenol blue front reached the bottom of the gel.
(2.2)40.0%ethanol,10.0%acetic acid fixed2times, each time15min.30.0%ethanol,0.2%Na2S2O3,6.8%sodium acetate-sensitized for30min. Following distilled water washed three times, each time5min.2.50‰AgNO3staining20min. Following distilled water washed two times, each time1min.2.5%Na2CO3,0.04‰formaldehyde to develop an image until protein spots clear. Then we immediately added the5.0%acetic acid to terminate for10min, following distilled water washed three times, each time5min. At last, we used30.0%ethanol and4.6%glycerol to preserve.
(2.3) the gel was scanned through the UMAX PowerLook1100scanner to obtain2-DE gel image, then using Melanie4image analysis software to analyse the image and produce differentially expressed protein spots data.
(3) To screen and separate the differentially expressed proteins between patients with bladder cancer and normal controls by two-dimensional fluorescent differential gel electrophoresis
(3.1) Protein isolated from the pooled urine samples were labeled with cyanine dyes according to the manufacturer's instructions.50μg of urine protein samples from bladder cancer and control group were minimally labeled with400pmol of Cy3and Cy5fluorescent dyes, respectively. To assess the reproducibility and statistical inferences, an internal standard pool was labeled with Cy2. The internal standard pool was generated by combining equal amounts of extracts from urinary samples of all neoplastic and control group subjects. The labeling reactions were carried out on ice for30min protecting from light, and then quenched with1ml of10mM lysine for10min. All three labeled samples were mixed and resolved in one gel.
(3.2) The method of the resting isoelectric focusing was similar to2-DE
(3.3) The three gels were visualized with a Typhoon9410scanner at the excitation emission of488/520nm (Cy2),532/580nm (Cy3) and633/670nm (Cy5), respectively. The images were analyzed with the DeCyder5.0software. Its differential in-gel analysis (DIA) module was used for pairwise comparisons of each sample with the internal standard within each gel by calculating the normalized spots volumes. The DeCyder biological variation analysis (BVA) module was used to calculate average abundance changes for each spot across the different spot maps. The spots whose ratios of Cy5/Cy2and Cy3/Cy2were up-or down-regulated equal or greater than2-fold were considered for further analysis
(3.4) In addition, another strip was performed in parallel as a preparative gel for spots picking as marked in2D-DIGE. Noticeably,1000mg of proteins were loaded onto the IPG strip and the gel was stained with Coomassie brilliant blue. The differentially expressed protein spots were cut from the Coomassie stained gels, and then identified by MALDI-FOF/TOF-MS. Briefly, Protein spots from gels were digested with trypsin solution, and the peptide mixtures was analyzed. A list of the corrected mass peaks was the peptide mass fingerprinting (PMF). The search was restricted to the Homo sapiens subsets of the sequences in the Swiss-Prot and NCBI nonredundant protein sequence databases.
3. GC was selected for further investigation after bioinformatics analysis
We conducted the bioinformatics research to study to explore the biological processes participated of thosed identified proteins, protein-protein interactions and protein-specific structures and functions, in order to select the candidate proteins for further study by those tools, such as:SWISS-PROT, String and Pubgene.
4. The expression of candidate proteins GC in the urine samples form healthy controls and patients with bladder cancer
(1) For western blot analyses, the samples involved two low grade and two high grade non-invasive papillary urothelial carcinoma, four infiltrating urothelial carcinoma and eight controls.30μg prepared proteins from urine and tissue were electrophoresed respectively on a12%SDS polyacrylamide gel and then transferred onto Polyvinylidene Xuoride (PVDF) membranes. The membranes were blocked in a solution of TBS containing5%nonfat milk powder and0.1%Tween-20for1h at room temperature and then incubated overnight at4℃with the primary antibody and rabbit monoclonal primary antibody against human β-actin. After washing with TBS-T for three times, the membranes were incubated with the secondary antibody dilution at room temperature for1h. The proteins were detected using an enhanced chemiluminescence detection system.
(2) ELISA was used to quantify urinary GC levels in91patients with bladder cancer,11benign bladder damage cases and42healthy controls. According to the criteria of World Health Organization classification of tumours,68cases were non-invasive papillary urothelial carcinoma (38were low grade and30were high) and23were infiltrating urothelial carcinoma.
5. The analysis of GC level with clinical data
To explore whether there was any correlation between the GC expression and features of bladder cancer such as age, gender, hematuria, recurrence and metastasis and classification of pathology. Receiving operating curve (ROC) analyses were used to define the optimal diagnostic cut-off value by estimating the sensitivity versus its false-positive rate at optimal cutoffs
6. Expression of GC in normal and tumour bladder tissue
(1) For western blot analyses, Tissue specimens were immediately frozen in liquid nitrogen, then ground to powder and homogenized in lysis buffer. The mixture was placed on a shaker at4℃for1h, and then followed by centrifugation. The supernatant was used for further analysis after measuring the protein concentrations. Then, the protein was used for western blotting.
(2) For Immunohistochemical analysis, There were eight low grade and eight high grade non-invasive papillary urothelial carcinoma, ten infiltrating urothelial carcinoma and ten normal controls in immunohistochemistry. The samples of normal controls were the normal-adjacent to-cancer tissues. Paraffin sections was dropped the wax then put into the water. The sections were incubated in3%hydrogen peroxide to quench the endogenous peroxidase activity and washed in PBS for three times. Plus the primary antibody conjugated with biotin (Biotin) at37℃for30minutes, then put in the wet box. Following PBS washed three times, then plus enzyme labeled avidin (Avidin) at37℃for30. PBS washed three times, following DAB chromogenic and washed in the running water. The sections was stained, dehydration and mounted at last.
Results
1. With the improvement of optimization steps of repeated ultrafiltration and desalination to sample, we could obtain high-resolution, reproducible two-dimensional electrophoresis maps from human urine samples.
2. A representative DIGE image from bladder cancer versus control group is acquired., a total of24differential protein spots whose volumes changed by or over2-fold were selected for further identification. Sixteen differentially expressed proteins were identified. The up-regulated proteins in bladder cancer were ALB, GC, HP, FGB, APOA1, RBP4and SECTM1, and the other nine proteins were down-regulated. They were UMOD,KNG1, AMY1A, AMY2A, ITIH4, AMBP, HSPG2, CST5and MASP2.
3. The analysis of Pubgene and String indicated that GC was predicted to play important roles in the bioprocesses of growth, secretion, induction, pathogenesis, signal transduction, digestion, translation, apoptosis and death. Furthermore, GC may tightly correlate with the other identified proteins.
4. Western blotting results demonstrated that GC was significantly upregulated in bladder cancer cases in comparison to controls, consistent with our DIGE results.
5. The expression level of GC was significantly higher in bladder cancer tissue in comparison to normal control bladder tissue. GC protein was detected, apparently in the cytoplasmic compartments of normal bladder transitional cells and cancerour cells by immunohisochemical staning of bladder sections.
6. GC-Cr was significantly elevated in patients with bladder cancer compared to controls and benign cases (1013.70±851.25versus99.34±55.87,105.32±47.81ng/mg, respectively, P<0.05). The urinary GC concentration was significantly higher in infiltrating urothelial carcinoma cases than in cases with low grade and high grade non-invasive papillary urothelial carcinoma (1906.69±840.86versus472.92±348.02and1014.06±753.16ng/mg, P<0.05).7. The optimum cut-off value was161.086ng/mg with92.31%sensitivity and83.02%specificity for diagnosis of bladder cancer. ROC analyses rendered a cut-off value with1407.481ng/mg corresponding to82.61%sensitivity and88.24%specificity d to distinguish infiltrating urothelial carcinoma form bladder cancer patients.
Conclusion
1. Establishing stability and good reproducibility human urine2-DE techniques and methods in our study, which laid a foundation for further bladder cancer research on urine proteme.
2. Comparing the urine proteome map of bladder cancer compared with normal controls by2D-DIGE.
3. Sixteen differentially expressed proteins were identified. The up-regulated proteins in bladder cancer were ALB, GC, HP, FGB, APOA1, RBP4and SECTM1, and the other nine proteins were down-regulated. They were UMOD, KNG1, AMY1A, AMY2A, ITIH4, AMBP, HSPG2, CST5and MASP2.
4. GC was selected for further investigation after bioinformatics analysis.
5. The expression level of GC was significantly higher in bladder cancer tissue in comparison to normal control bladder tissue. GC protein was detected, apparently in the cytoplasmic compartments of cells.
6. GC is significantly elevated in bladder cancer, and is positively associated with the pathological classification of bladder cancer
7. Urinary GC may be a potential biomarker for the early diagnosis and effective surveillance of bladder cancer.
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