卵巢癌早期诊断标志物的蛋白组学分析及GRP siRNA对卵巢癌细胞ES2生物学效应的研究
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摘要
1.研究背景
卵巢癌是目前全球死亡率最高的妇科恶性肿瘤。究其原因,是因为卵巢的解剖位置隐匿,早期卵巢癌常缺乏临床症状,而造成患者就诊时往往到了晚期,晚期病人的治疗过程中,常发生耐药和复发,5年生存率只有15-20%,与晚期相比,早期癌的5年生存率高达80～90%,可见提高卵巢癌的5年生存率的关键在于发现更多的早期病人。由于缺乏相应的高度特异性的标志物,卵巢癌的早期诊断仍是其研究的一个瓶颈问题,因此,卵巢癌的早期诊断仍是目前研究的热点。目前临床上多采用阴道B超结合CA125早期诊断卵巢癌,但是尽管这样也只能发现25%左右的早期病例,诊断性腹腔镜能够发现100%的卵巢癌病人,但是其有创伤性和昂贵的价格限制了其在临床上的广泛应用。卵巢癌早期诊断标志物的研究中,多数学者采用蛋白质组学方法进行筛选,其最大的优势在于能够从整体水平把握整个疾病的病理生理状态,传统的蛋白组学方法有2-DE、多维凝胶电泳,但是这些方法的可重复性较低且费时费力,近年来激光解析电离飞行时间质谱和基质辅助电离飞行时间质谱两大技术飞速发展,尤其是前者目前多用于血清标志物的筛选,但其可重复性仍然较差,随着研究的日益成熟,一些学者采用磁珠结合MALDI技术进行肿瘤标志物筛选的研究时发现该方法的可重复性较高,认为这与磁珠表面的疏水特性有关。
较早的蛋白组学研究多采用血清标本来进行疾病蛋白质的筛选,近年来随着蛋白质分离技术、质谱分析技术和蛋白质数据库生物信息学技术的迅猛发展,唾液、胆汁、腹水、囊液等体液也被用来进行蛋白质组学的研究,目前多数研究仍集中在血清标志物筛选上,但血清蛋白来自全身,器官特异性较低,很难从大多数无症状人群中捕获尽可能多的真正患病的个体,相比较而言,各种体液蛋白质组学研究所筛选的标志物具有特异性高的优势。最近,虽有将卵巢癌患者的囊液和腹水用来筛选早期诊断标志物的研究,但是将各个期别的卵巢癌作为一个研究组与对照组进行对照研究,势必会遗漏一些早期病变的标志物。
很显然,将卵巢癌分为早期组和晚期组,并将血清、腹水和囊液相结合用以筛选早期诊断的标志物,将会大大提高所筛选标志物的特异性和敏感性。因此本研究采用基于磁性微球的MALDI-TOF-MS、免疫组化、酶联免疫吸附试验技术,首先将卵巢癌患者分为早期组和晚期组分别与两个对照组进行比对研究,进一步在腹水和囊液中筛选卵巢癌相关侯选蛋白和早期诊断标志物,从蛋白水平探讨卵巢癌的血清和组织分子变化特征和规律,并采用小干扰RNA技术初步探讨筛选标志物的生物学功能,加深对卵巢癌癌变机制的了解,并为进一步发掘卵巢癌早期发现的分子指标和手段提供重要的理论基础和信息。
2.材料与方法
蛋白质组学实验：
血清：训练集血清：卵巢癌患者30例(其中Ⅰ-Ⅱ期11例,Ⅲ～Ⅳ期19例,浆液性23例,粘液性6例,内膜样癌1例；<50岁9例,>50岁21例,家族史阳性10例,家族史阴性20例)、良性卵巢肿瘤10例(其中浆液性8例,粘液性2例)和正常卵巢者13例的血清和配对组织,5例卵巢癌患者的腹水和囊液,以上血清为训练集样本,验证集血清：卵巢癌8例(其中Ⅰ-Ⅱ期2例,Ⅲ-Ⅳ期6例)、5例卵巢良性肿瘤(其中浆液性4例,粘液性1例)和5例正常卵巢者的血清作为验证集样本。
免疫组织化学实验：
卵巢癌和正常卵巢组织为与以上训练集血清配对的组织共43例,良性卵巢肿瘤组织20例(与训练集血清配对组织10例,与验证集血清配对组织5例和额外收集组织5例)。
酶联免疫吸附实验：
卵巢癌和正常卵巢组为训练集加验证集血清共56例,良性卵巢肿瘤组25例(除训练集10例外,额外收集10例也作为训练集和验证集5例)。
小干扰RNA实验：
细胞：人卵巢癌细胞系ES2细胞
在2006年7月至2009年1月收集术前日清晨空腹血5ml,离心后-80℃保存待用。组织均取自术中,10%福尔马林固定,石蜡包埋并以蜡块室温保存。所有病人术前均排除内外科合并症和肾功能不全,未经过放化疗和免疫治疗。术中收集腹水和囊液标本并避免被血液污染。
取5μl血清样品,加入弱阳离子磁珠WCX (Bruker Daltonics, Leipzig Germany)混悬液SPE-CM2μl以及SPE-CB95μl,磁珠结合并贴壁后移去上清,再加入SPE-CW100μL,反复重复操作两次,得到制备好的血清样品。腹水和囊液的上样量分别为10μL和2.5μL,操作步骤和血清样品制备相同。
将1μ1含蛋白质的洗脱液与10μl基质(0.3%的-α-氰基-4-羟基肉桂酸,HCCA)混合,取1μl点样于Anchorchip靶板上,将耙板放入Microflex质谱仪(Bruker Daltonics),用标准品(Clinprot standard)校正仪器后检测样品,获得不同质荷比的蛋白质峰构成的质谱图。在血清、腹水和囊液中筛选共同升高的多肽峰。然后在http://us.expasy.org/tools/tagident. html数据库中检索与之匹配的多肽。根据既往文献报道,甄别筛选出候选多肽进行下一步验证。
采用免疫组化SP法检测卵巢癌组织、良性卵巢肿瘤组织和正常卵巢组织的相应多肽的表达。GRP抗体(rabbit polyclonal, MAB35841 R&D Co, Ameraican)和二抗、DAB试剂盒均购自上海越研生物工程公司。实验按照试剂盒说明书进行。
采用酶联免疫吸附试验检测卵巢癌组、良性卵巢肿瘤组和正常卵巢组的血清相应多肽的水平差异。GRP ELISA试剂盒(QC01-1, R & D Co, American)购自上海越研生物工程公司。Pro-GRP试剂盒(H23517,Ⅵ, American)。实验按照试剂盒说明书进行
构建针对GRP的发夹样siRNA真核表达载体,人工合成GRPsiRNA的寡核苷酸链,导入p-Genesil-1.1载体(p-Genesil-1.1载体购自武汉晶赛生物技术工程公司),采用脂质体转染入人卵巢癌细胞系ES2细胞中,采用免疫印迹法(Western bloting)和逆转录聚合酶链式反应(RT-PCR)技术检测转染后GRPmRNA和蛋白表达变化,采用细胞计数法检测各组细胞增殖情况；流式细胞术观察被转染细胞的凋亡情况；transwell小室观察细胞被转染前后的穿膜侵袭力。
采用ClinProtTM(Bruker Daltonics Company)的内置软件Flex Analysis 3.0收集所有样品的质谱峰,并分析单个峰的强度。采用Top Hat和Savitsky Golay系统进行平滑、去噪和归一化处理后采用遗传算法对多肽峰进行量化。采用ClinProtTM软件(Ver.2.2; Bruker Daltonics)将所有信噪比(S／N)>5的1000-10000Da范围内的信号进行处理。通过Kruskal-Wallis检验的P值评价每个差异峰的在不同组别的表达强度差异。采用线性支持向量机(SVM)区分各组的数据并建立诊断模型,以留一法分析诊断模型的诊断敏感度和特异度。筛选出的多肽峰的诊断意义用样品分布图和均值标准差图来表示。免疫组化的酶联免疫吸附试验的结果分别用χ2检验和方差分析(q-检验)、Kruskal-Wallis法进行分析。分别采用Spearman相关性分析评价卵巢癌组的免疫组化和ELISA结果一致性和Pearson相关性分析检验GRP和pro-GRP这两个血清指标的相关性,计算ELISA结果的95%的可信区间和敏感度、特异度、阳性预测值和阴性预测值。以Pearson相关系数表示差异多肽峰和血清多肽水平的相关性。各组的转染前后的蛋白、基因表达水平的变化和增殖、侵袭力变化采用两样本t检验分析。采用统计学软件SPSS13.0数据包进行统计分析。3结果
卵巢癌组、良性组和正常组三组比较分析：良性与正常组相比无差异峰。与良性组相比,早、晚期卵巢癌组共同升高的多肽峰有2881和2897Da(P<0.05)；与正常组相比,早、晚期卵巢癌组共同升高的多肽峰有2881,2897和4466Da(P<0.05)。在4例卵巢癌患者的腹水和囊液中也发现2881和2897Da多肽峰均值较高。选择早晚期卵巢癌患者血清、腹水和囊液中共同升高的2881和2897Da差异峰采用GC算法建模,经留一法验证,诊断卵巢癌的特异度为100%,敏感度为100%,盲法检测的诊断特异度为100%,敏感度为87.5%。
年龄<50岁和>50岁卵巢癌组与正常卵巢组相比无明显差异多肽峰(P均>0.05),家族史阳性与阴性的卵巢癌患者,血清多肽谱与正常卵巢组相比,仅有两个下调的多肽峰2953和1466Da一致(P<0.05),其余差异多肽峰均不同。
在http://us.expasy.org/tools/tagident. html数据库中检索与2881和2897Da匹配的多肽,依据统计学分析及文献检索结果,在9种卵巢癌候选相关差异多肽中,仅胃泌素释放肽(Gastrin-releasing peptide,GRP)在既往报道中较多提示与肿瘤密切相关,其余均未见报道与肿瘤有关。
免疫组化结果显示,与良性(30%)和正常组(0%)相比,GRP在卵巢癌组织中显著高表达(早期：72.73%；晚期：78.95%),差异有显著性(χ2=24.599,P=0.00)。ELISA结果显示GRP在卵巢癌血清中显著高于良性和正常卵巢组(P<0.05)。Pearson相关性检验显示,卵巢癌患者的血清2881和2897Da差异多肽峰均值和标准差与GRP血清水平有明显相关性(r=0.92；r=0.88；P均<0.0001)。此外,卵巢癌组织和血清中的GRP呈正相关且一致性较高(r=0.809,P=0.000),GRP与其前体pro-GRP在训练集人群中有明显的相关性(r=0.90,P<0.001),且pro-GRP在训练集人群血清中的水平显著高于另外两组(χ2=41.81,P=0.00),其作为诊断指标的ROC面积为d.974(z=27.88,P<0.001),cut-off值为89.2851,用于诊断卵巢癌的灵敏度为100%,特异度为90.91%,Youden指数为0.9091。对验证集人群进行验证的灵敏度为93.33%,特异度为80%,Youden指数为0.7333。
成功构建人GRP的发夹样siRNA真核表达载体GRPsiRNA,转染ES2细胞后,其增殖能力明显下降(P<0.05)；表达GRPmRNA和蛋白明显降低并且凋亡率显著下降；transwell小室显示其穿膜侵袭能力明显下降(P<0.05)。
4结论
4.1在早、晚期卵巢癌患者的血清中共同升高并在腹水和囊液也发现均值较高的2881和2897Da差异多肽峰,是高度特异和敏感的卵巢癌早期诊断候选标志物。家族史阳性和阴性的卵巢癌患者发病机理存在不同。
4.2 GRP可能是与2881和2897Da相匹配的多肽,其前体pro-GRP对卵巢癌具有较高的早期诊断价值。
4.3GRPsiRNA构建成功并转染ES2细胞可显著降低其增殖能力和穿膜侵袭力,并促进其凋亡
1. Background
Obviously, OC is the most lethal gynecological cancer. It often eludes the clinician, due to its insidious localization in the pelvis and lacking of symptoms and signs during early stages (stageⅠ/Ⅱ). This is the reason for that nearly 70% patients are not diagnosed with OC until the advanced stages (stageⅢ/Ⅳ). The five-year overall survival of advanced stage is no more than 20%, compared to the 90% of early stage cases. Therefore, increasing the number of women diagnosed in early stage should have a direct effect on the mortality and economics of this cancer.
Lacking of highly specific biomarker remains the main bottleneck problem in the study of early detection of OC. Proteomic can afford a full view of disease and has been used abroadly in research of many diseases. Although 2-dimensional gel electrophoresis and multi-dimensional liquid chromatography after mass spectrometry (MS) analysis seemed a somewhat economical, these techniques were also laborious and time-consuming. Recently, the discovery of biomarkers in body fluids has been advanced by the introduction of MS based screening methods such as Matrix-assisted Laser Desorption/ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and a protein chip coupled with Surface enhanced Laser Desorption/Inionation Time of Flight Mass Spectrometry (SELDI-TOF-MS). The first clinical investigations using SELDI-TOF-MS in different cancer types revealed high diagnostic sensitivities and specificities.SELDI-TOF-MS has been developed to facilitate protein profiling of complex biological mixtures, with high efficacy discovery of cancer biomarkers in serum or plasma. However, different patterns for the same type of cancers have been identified by different individual groups using the same types of biological specimens and analytical platforms of SELDI-TOF-MS. These discrepancies could be attributed to poorly define analytical protocols, which were not established reproducibility. Based on magnetic beads integrated with MALDI-TOF-MS analyzing, serum peptides were fractionated and concentrated on surface-modified targets with specific protein-capture properties by some scholars and they attributed the high reproducibility to the hydrophobic surface of magneatic beads.
Following with the development of the technique of proteomic, many kinds of body fluid such as bile, saliva, ascites have been used for the screening of tumor biomarkers. Many studies focus on secreening the biomarker in serum of patients with OC by proteomic. However, the proteins and peptides in the serum came from all over the body, which made the specificity and sensitivity of these serum biomarkers insufficient to capture most of the patients in early stage from the large scale of women without any symptoms. Furthermore, taking patients with OC in different stages as a whole group would undoubtly leaving out some biomarker of early stage.
Evidently, combining the serum, ascite and cyst fluid of OC and devide the OC group into early stage and advanced stage groups will enhance the specificity of the biomarker of OC. In the present study, MALDI-TOF-MS integrating with the megneatic beads was used to screening the biomarker of early detection of OC. Immunohistochemistry and enzyme linked immuno adsorbing were used to validated the suspected peptide. Small interfering RNA technique was used to understand the biological effects of suspected peptides on ovarian carcinoma cell line.
2. Materials and methods Proteomic: training set:A total of 30 consecutive patients with OC (11 in early stages and 19 in advanced stage; 23 of serous and 6 of mucinous and one of endometrioid cancinoma; nine with age<50years old and 21 with>50 years old; 10 with positive familial history and 20 with negative familial history.),10 with benign ovarian tumors (10 of serous and 2 of mucinous) and 13 age-matching patients without cancer and tumor collected from the Department of Gynecology, the First and Second Affiliated Hospitals of Zhengzhou University, from July 2006 to January 2009, were recruited in this study. Testing set:8 cases of OC (6 serous and 2 mucinous),5 cases of benign ovarian tumor (4 serous and 1 mucinous) and 5 cases of normal ovarian. The ascites and cyst fluid samples were also collected by the same method during the operation.
IHC:The matching tissues of these 53 cases of training set, testing set and another 10 cases of benign tissues were selected for IHC.
ELISA:All of the cases of training set and testing set were identical to the cases of proteomic except for additional 10 cases of benign patients without matching tissues were also used for training set.
Small interfere RNA:
ES2 ovarian cancer cell line was used to peform the study of gene silencing of the target gene.
All patients were collected the venous blood for 5ml in the morning the day before the operation and stored at -80℃after centrifuging. Ascites and cyst fluid samples further obtained from five patients were dealed with the same method. No treatment was implemented on all patients before surgery. All of the participants were identified as normal renal function.
All of the tissues matching with the serum were collected at the beginning of the operation and were disposed by paraffin after being fixed with 10% formalin.
2μl Suspention of Weak cation beads (WCX) SPE-CM,95μl of SPE-CB and 100μL of SPE-CW was added into 5μl of serum sample. The prepared samples were obtained after several binging and elution. The ascites and cyst fluid samples were 10μL and 2.5μL respectively for the same preparing method.
1μl of eluent with peptides was mixed with 10μl of matrix (0.3%HCCA) and then 1μl of mixture was applicated onto the Anchorchip. Put the buck sracper into Microflex mass spectrometer (Bruker Daltonics) after calibrated by the standard sample (Clinprot standard). Massspectrograms of peptides peaks with different m/z were obtained after detecting the samples. Then the common elevated peaks in the serum, ascites and cyst fluid were selected for the candidated peptide peaks. At last, the matching peptides were searching in the http://us.expasy.org/tools/tagident. html protein data bank. After retrievaling from prvious data, the deducted cnadidated peptides were selected for next validation.
Immunostaining was performed with the antibodies of the GRP (rabbit polyclonal, MAB35841 R&D Co, Ameraican) and IgG fluorescence Secondary Antibodies (surpported by Yueyan Biotech Co. Shanghai). According to the manufacture's protocol, a standard streptavidin-biotin-peroxidase method was used.
Serum level of GRP was detected by gastrin-releasing peptide ELISA kit (QC01-1, R & D Co, American). The reactions were done by following the manufacture's protocol.
A GRPsiRNA targetting the human GRPmRNA common sequence was constructed and inserted onto the Bam Hi-HindⅡlinearized p-genesil-1 vector. After transfected the plasmid into the ES2 cell, level of GRPmRNA and protein were detected by RT-PCR and Western bloting after the transfection. The proliferation, apoptosis and capability of migration and invasiveness were evaluated by cell dounting, flow cytometry and transwell.
The build-in ClinProtTM software (Bruker Daltonics Company) and FlexAnalysis 3.0 were used to compile the peaks across the spectra and analyze the intensity of single peak. All signals with a signal-to-noise (S/N) ratio>5 in a mass range of 1000-10000 Da were recorded with the ClinProtTM software (Ver.2.2; Bruker Daltonics). A linear SVM classifier was used to distinguish between the different groups of data. The capability of each peak in dstinguishing different groups of data was estimated by the P value of Kruskal-Wallis test. The combinations of the selected peaks was analyzed by the leave-one-out cross-validation. The results of IHC and ELISA were analyzed byχ2 test, q-test and Kruskal-Wallis. Spearman correlations and Pearson correlation coefficient analysis was used to get the relevance of the IHC and ELISA and the GRP and pro-GRP level. Two sample t-test was used to analysize the different level of proliferation, apoptosis, capability of migration, mRNA and protein of GRP before and after transfection in the cell. These data analysis were completed by analysis software SPSS 13.0.
3.Results
MALDI-TOF-MS was used to profile the mass spectral patterns of serum samples from three groups and found that there were no different peaks between benign and normal groups. The common elevated peaks in early and advanced stage of OC were 2881 and 2897Da compared with the normal group (P<0.05). The common elevated peaks in early and advanced stage of OC were 2881,2897 and 4466Da (P<0.05). It was further found that the 2881 and 2897Da peaks were also elevated in the ascite and cyst fluid samples. The pattern of two combined peaks had a specificity of 100% and a sensitivity of 100%, as evaluated by leave-one-out crossvalidation. The remaining 18 serum samples were analyzed as a blind test set. The specificity and sensitivity of the blind test were 100% and 87.5%, respectively.
Compared with normal ovarian group, there were no significant defferential peaks in below and above 50 years old groups (P all>0.05). Compared with normal ovarian group, only 2953 and1466Da peaks were found downregulated in the positive and negative familial history groups (P<0.05) while other differential peaks were thoroughly different.
The candidate peptides matching with 2881 and 2897Da were searching in the http://us.expasy.org/tools/tagident. html peptide data bank. According to the statistic and the reported data, only Gastrin-releasing peptide (GRP) were found closely related to many kinds of cancers among the nine kinds of candidate peptide of OC searching by us.
The positive expression of GRP in tissues of OC were higher than the benign tumor and normal ovary. The expressions of GRP in the normal group were all low to negative expression (χ2= 24.599, P= 0.00). The higher serum level of GRP in OC than benign and normal groups was verified by ELISA (P<0.05, by q-test). There was a high correlation between level of Gastrin-releasing peptide in tissue and serum (r=0.809, P=0.000). The correlations of the serum level of 2881,2897Da peaks and serum level of GRP in three groups were all high. There was an obviously coefficient between GRP and pro-GRP in the training set (r=0.90, P<0.001). Pro-GRP level in serum of training set significantly higher than the benign and normal groups(χ2= 41.81, P=0.00). The area below ROC curve was 0.974 when the proGRP was used for diagnostic marker (z=27.88, P<0.001). The cut-off-value was 89.2851, which had a sensitivity of 100% and a specificity of 90.91%(Youden index was 0.9091) in training set and a sensitivity of 93.33% and a specificity of 80%(Youden index was 0. 7333) in testing set.
Cells stably expressing GRPsiRNA were got by G418 screening after transfection. The proliferation and capability of migration and invasiveness of ES2 cell were significantly decreased (P<0.05) after the transfection accompanied by the down-regulation of the mRNA and protein level of GRP (P<0.05). As a result, the apoptosis of ES2 cell was also activated by the transfection (P<0.05).
4. conclusion
4.1 The 2881 and 2897 Da peaks are highly specific and sensitive serum biomarkers for early detection of OC. The mechanism of the carcinogenesis of hereditary and nonhereditary OC might be partly different
4.2 Gastrin-releasing peptide might be the candidated peptide matching with the 2881 and 2897Da peaks. Pro-GRP had a high diagnostic value in detecting OC with early stage.
4.3. ES2 cell transfected by the GRPsiRNA show a decreased capability of proliferation, migration and invasiveness as well as the activation of the apoptosis. GRP might be a potential target for treatment of OC.
卵巢癌是目前全球死亡率最高的妇科恶性肿瘤。究其原因,是因为卵巢的解剖位置隐匿,早期卵巢癌常缺乏临床症状,而造成患者就诊时往往到了晚期,晚期病人的治疗过程中,常发生耐药和复发,5年生存率只有15-20%,与晚期相比,早期癌的5年生存率高达80～90%,可见提高卵巢癌的5年生存率的关键在于发现更多的早期病人。由于缺乏相应的高度特异性的标志物,卵巢癌的早期诊断仍是其研究的一个瓶颈问题,因此,卵巢癌的早期诊断仍是目前研究的热点。目前临床上多采用阴道B超结合CA125早期诊断卵巢癌,但是尽管这样也只能发现25%左右的早期病例,诊断性腹腔镜能够发现100%的卵巢癌病人,但是其有创伤性和昂贵的价格限制了其在临床上的广泛应用。卵巢癌早期诊断标志物的研究中,多数学者采用蛋白质组学方法进行筛选,其最大的优势在于能够从整体水平把握整个疾病的病理生理状态,传统的蛋白组学方法有2-DE、多维凝胶电泳,但是这些方法的可重复性较低且费时费力,近年来激光解析电离飞行时间质谱和基质辅助电离飞行时间质谱两大技术飞速发展,尤其是前者目前多用于血清标志物的筛选,但其可重复性仍然较差,随着研究的日益成熟,一些学者采用磁珠结合MALDI技术进行肿瘤标志物筛选的研究时发现该方法的可重复性较高,认为这与磁珠表面的疏水特性有关。
较早的蛋白组学研究多采用血清标本来进行疾病蛋白质的筛选,近年来随着蛋白质分离技术、质谱分析技术和蛋白质数据库生物信息学技术的迅猛发展,唾液、胆汁、腹水、囊液等体液也被用来进行蛋白质组学的研究,目前多数研究仍集中在血清标志物筛选上,但血清蛋白来自全身,器官特异性较低,很难从大多数无症状人群中捕获尽可能多的真正患病的个体,相比较而言,各种体液蛋白质组学研究所筛选的标志物具有特异性高的优势。最近,虽有将卵巢癌患者的囊液和腹水用来筛选早期诊断标志物的研究,但是将各个期别的卵巢癌作为一个研究组与对照组进行对照研究,势必会遗漏一些早期病变的标志物。
很显然,将卵巢癌分为早期组和晚期组,并将血清、腹水和囊液相结合用以筛选早期诊断的标志物,将会大大提高所筛选标志物的特异性和敏感性。因此本研究采用基于磁性微球的MALDI-TOF-MS、免疫组化、酶联免疫吸附试验技术,首先将卵巢癌患者分为早期组和晚期组分别与两个对照组进行比对研究,进一步在腹水和囊液中筛选卵巢癌相关侯选蛋白和早期诊断标志物,从蛋白水平探讨卵巢癌的血清和组织分子变化特征和规律,并采用小干扰RNA技术初步探讨筛选标志物的生物学功能,加深对卵巢癌癌变机制的了解,并为进一步发掘卵巢癌早期发现的分子指标和手段提供重要的理论基础和信息。
2.材料与方法
蛋白质组学实验：
血清：训练集血清：卵巢癌患者30例(其中Ⅰ-Ⅱ期11例,Ⅲ～Ⅳ期19例,浆液性23例,粘液性6例,内膜样癌1例；<50岁9例,>50岁21例,家族史阳性10例,家族史阴性20例)、良性卵巢肿瘤10例(其中浆液性8例,粘液性2例)和正常卵巢者13例的血清和配对组织,5例卵巢癌患者的腹水和囊液,以上血清为训练集样本,验证集血清：卵巢癌8例(其中Ⅰ-Ⅱ期2例,Ⅲ-Ⅳ期6例)、5例卵巢良性肿瘤(其中浆液性4例,粘液性1例)和5例正常卵巢者的血清作为验证集样本。
免疫组织化学实验：
卵巢癌和正常卵巢组织为与以上训练集血清配对的组织共43例,良性卵巢肿瘤组织20例(与训练集血清配对组织10例,与验证集血清配对组织5例和额外收集组织5例)。
酶联免疫吸附实验：
卵巢癌和正常卵巢组为训练集加验证集血清共56例,良性卵巢肿瘤组25例(除训练集10例外,额外收集10例也作为训练集和验证集5例)。
小干扰RNA实验：
细胞：人卵巢癌细胞系ES2细胞
在2006年7月至2009年1月收集术前日清晨空腹血5ml,离心后-80℃保存待用。组织均取自术中,10%福尔马林固定,石蜡包埋并以蜡块室温保存。所有病人术前均排除内外科合并症和肾功能不全,未经过放化疗和免疫治疗。术中收集腹水和囊液标本并避免被血液污染。
取5μl血清样品,加入弱阳离子磁珠WCX (Bruker Daltonics, Leipzig Germany)混悬液SPE-CM2μl以及SPE-CB95μl,磁珠结合并贴壁后移去上清,再加入SPE-CW100μL,反复重复操作两次,得到制备好的血清样品。腹水和囊液的上样量分别为10μL和2.5μL,操作步骤和血清样品制备相同。
将1μ1含蛋白质的洗脱液与10μl基质(0.3%的-α-氰基-4-羟基肉桂酸,HCCA)混合,取1μl点样于Anchorchip靶板上,将耙板放入Microflex质谱仪(Bruker Daltonics),用标准品(Clinprot standard)校正仪器后检测样品,获得不同质荷比的蛋白质峰构成的质谱图。在血清、腹水和囊液中筛选共同升高的多肽峰。然后在http://us.expasy.org/tools/tagident. html数据库中检索与之匹配的多肽。根据既往文献报道,甄别筛选出候选多肽进行下一步验证。
采用免疫组化SP法检测卵巢癌组织、良性卵巢肿瘤组织和正常卵巢组织的相应多肽的表达。GRP抗体(rabbit polyclonal, MAB35841 R&D Co, Ameraican)和二抗、DAB试剂盒均购自上海越研生物工程公司。实验按照试剂盒说明书进行。
采用酶联免疫吸附试验检测卵巢癌组、良性卵巢肿瘤组和正常卵巢组的血清相应多肽的水平差异。GRP ELISA试剂盒(QC01-1, R & D Co, American)购自上海越研生物工程公司。Pro-GRP试剂盒(H23517,Ⅵ, American)。实验按照试剂盒说明书进行
构建针对GRP的发夹样siRNA真核表达载体,人工合成GRPsiRNA的寡核苷酸链,导入p-Genesil-1.1载体(p-Genesil-1.1载体购自武汉晶赛生物技术工程公司),采用脂质体转染入人卵巢癌细胞系ES2细胞中,采用免疫印迹法(Western bloting)和逆转录聚合酶链式反应(RT-PCR)技术检测转染后GRPmRNA和蛋白表达变化,采用细胞计数法检测各组细胞增殖情况；流式细胞术观察被转染细胞的凋亡情况；transwell小室观察细胞被转染前后的穿膜侵袭力。
采用ClinProtTM(Bruker Daltonics Company)的内置软件Flex Analysis 3.0收集所有样品的质谱峰,并分析单个峰的强度。采用Top Hat和Savitsky Golay系统进行平滑、去噪和归一化处理后采用遗传算法对多肽峰进行量化。采用ClinProtTM软件(Ver.2.2; Bruker Daltonics)将所有信噪比(S／N)>5的1000-10000Da范围内的信号进行处理。通过Kruskal-Wallis检验的P值评价每个差异峰的在不同组别的表达强度差异。采用线性支持向量机(SVM)区分各组的数据并建立诊断模型,以留一法分析诊断模型的诊断敏感度和特异度。筛选出的多肽峰的诊断意义用样品分布图和均值标准差图来表示。免疫组化的酶联免疫吸附试验的结果分别用χ2检验和方差分析(q-检验)、Kruskal-Wallis法进行分析。分别采用Spearman相关性分析评价卵巢癌组的免疫组化和ELISA结果一致性和Pearson相关性分析检验GRP和pro-GRP这两个血清指标的相关性,计算ELISA结果的95%的可信区间和敏感度、特异度、阳性预测值和阴性预测值。以Pearson相关系数表示差异多肽峰和血清多肽水平的相关性。各组的转染前后的蛋白、基因表达水平的变化和增殖、侵袭力变化采用两样本t检验分析。采用统计学软件SPSS13.0数据包进行统计分析。3结果
卵巢癌组、良性组和正常组三组比较分析：良性与正常组相比无差异峰。与良性组相比,早、晚期卵巢癌组共同升高的多肽峰有2881和2897Da(P<0.05)；与正常组相比,早、晚期卵巢癌组共同升高的多肽峰有2881,2897和4466Da(P<0.05)。在4例卵巢癌患者的腹水和囊液中也发现2881和2897Da多肽峰均值较高。选择早晚期卵巢癌患者血清、腹水和囊液中共同升高的2881和2897Da差异峰采用GC算法建模,经留一法验证,诊断卵巢癌的特异度为100%,敏感度为100%,盲法检测的诊断特异度为100%,敏感度为87.5%。
年龄<50岁和>50岁卵巢癌组与正常卵巢组相比无明显差异多肽峰(P均>0.05),家族史阳性与阴性的卵巢癌患者,血清多肽谱与正常卵巢组相比,仅有两个下调的多肽峰2953和1466Da一致(P<0.05),其余差异多肽峰均不同。
在http://us.expasy.org/tools/tagident. html数据库中检索与2881和2897Da匹配的多肽,依据统计学分析及文献检索结果,在9种卵巢癌候选相关差异多肽中,仅胃泌素释放肽(Gastrin-releasing peptide,GRP)在既往报道中较多提示与肿瘤密切相关,其余均未见报道与肿瘤有关。
免疫组化结果显示,与良性(30%)和正常组(0%)相比,GRP在卵巢癌组织中显著高表达(早期：72.73%；晚期：78.95%),差异有显著性(χ2=24.599,P=0.00)。ELISA结果显示GRP在卵巢癌血清中显著高于良性和正常卵巢组(P<0.05)。Pearson相关性检验显示,卵巢癌患者的血清2881和2897Da差异多肽峰均值和标准差与GRP血清水平有明显相关性(r=0.92；r=0.88；P均<0.0001)。此外,卵巢癌组织和血清中的GRP呈正相关且一致性较高(r=0.809,P=0.000),GRP与其前体pro-GRP在训练集人群中有明显的相关性(r=0.90,P<0.001),且pro-GRP在训练集人群血清中的水平显著高于另外两组(χ2=41.81,P=0.00),其作为诊断指标的ROC面积为d.974(z=27.88,P<0.001),cut-off值为89.2851,用于诊断卵巢癌的灵敏度为100%,特异度为90.91%,Youden指数为0.9091。对验证集人群进行验证的灵敏度为93.33%,特异度为80%,Youden指数为0.7333。
成功构建人GRP的发夹样siRNA真核表达载体GRPsiRNA,转染ES2细胞后,其增殖能力明显下降(P<0.05)；表达GRPmRNA和蛋白明显降低并且凋亡率显著下降；transwell小室显示其穿膜侵袭能力明显下降(P<0.05)。
4结论
4.1在早、晚期卵巢癌患者的血清中共同升高并在腹水和囊液也发现均值较高的2881和2897Da差异多肽峰,是高度特异和敏感的卵巢癌早期诊断候选标志物。家族史阳性和阴性的卵巢癌患者发病机理存在不同。
4.2 GRP可能是与2881和2897Da相匹配的多肽,其前体pro-GRP对卵巢癌具有较高的早期诊断价值。
4.3GRPsiRNA构建成功并转染ES2细胞可显著降低其增殖能力和穿膜侵袭力,并促进其凋亡
1. Background
Obviously, OC is the most lethal gynecological cancer. It often eludes the clinician, due to its insidious localization in the pelvis and lacking of symptoms and signs during early stages (stageⅠ/Ⅱ). This is the reason for that nearly 70% patients are not diagnosed with OC until the advanced stages (stageⅢ/Ⅳ). The five-year overall survival of advanced stage is no more than 20%, compared to the 90% of early stage cases. Therefore, increasing the number of women diagnosed in early stage should have a direct effect on the mortality and economics of this cancer.
Lacking of highly specific biomarker remains the main bottleneck problem in the study of early detection of OC. Proteomic can afford a full view of disease and has been used abroadly in research of many diseases. Although 2-dimensional gel electrophoresis and multi-dimensional liquid chromatography after mass spectrometry (MS) analysis seemed a somewhat economical, these techniques were also laborious and time-consuming. Recently, the discovery of biomarkers in body fluids has been advanced by the introduction of MS based screening methods such as Matrix-assisted Laser Desorption/ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and a protein chip coupled with Surface enhanced Laser Desorption/Inionation Time of Flight Mass Spectrometry (SELDI-TOF-MS). The first clinical investigations using SELDI-TOF-MS in different cancer types revealed high diagnostic sensitivities and specificities.SELDI-TOF-MS has been developed to facilitate protein profiling of complex biological mixtures, with high efficacy discovery of cancer biomarkers in serum or plasma. However, different patterns for the same type of cancers have been identified by different individual groups using the same types of biological specimens and analytical platforms of SELDI-TOF-MS. These discrepancies could be attributed to poorly define analytical protocols, which were not established reproducibility. Based on magnetic beads integrated with MALDI-TOF-MS analyzing, serum peptides were fractionated and concentrated on surface-modified targets with specific protein-capture properties by some scholars and they attributed the high reproducibility to the hydrophobic surface of magneatic beads.
Following with the development of the technique of proteomic, many kinds of body fluid such as bile, saliva, ascites have been used for the screening of tumor biomarkers. Many studies focus on secreening the biomarker in serum of patients with OC by proteomic. However, the proteins and peptides in the serum came from all over the body, which made the specificity and sensitivity of these serum biomarkers insufficient to capture most of the patients in early stage from the large scale of women without any symptoms. Furthermore, taking patients with OC in different stages as a whole group would undoubtly leaving out some biomarker of early stage.
Evidently, combining the serum, ascite and cyst fluid of OC and devide the OC group into early stage and advanced stage groups will enhance the specificity of the biomarker of OC. In the present study, MALDI-TOF-MS integrating with the megneatic beads was used to screening the biomarker of early detection of OC. Immunohistochemistry and enzyme linked immuno adsorbing were used to validated the suspected peptide. Small interfering RNA technique was used to understand the biological effects of suspected peptides on ovarian carcinoma cell line.
2. Materials and methods Proteomic: training set:A total of 30 consecutive patients with OC (11 in early stages and 19 in advanced stage; 23 of serous and 6 of mucinous and one of endometrioid cancinoma; nine with age<50years old and 21 with>50 years old; 10 with positive familial history and 20 with negative familial history.),10 with benign ovarian tumors (10 of serous and 2 of mucinous) and 13 age-matching patients without cancer and tumor collected from the Department of Gynecology, the First and Second Affiliated Hospitals of Zhengzhou University, from July 2006 to January 2009, were recruited in this study. Testing set:8 cases of OC (6 serous and 2 mucinous),5 cases of benign ovarian tumor (4 serous and 1 mucinous) and 5 cases of normal ovarian. The ascites and cyst fluid samples were also collected by the same method during the operation.
IHC:The matching tissues of these 53 cases of training set, testing set and another 10 cases of benign tissues were selected for IHC.
ELISA:All of the cases of training set and testing set were identical to the cases of proteomic except for additional 10 cases of benign patients without matching tissues were also used for training set.
Small interfere RNA:
ES2 ovarian cancer cell line was used to peform the study of gene silencing of the target gene.
All patients were collected the venous blood for 5ml in the morning the day before the operation and stored at -80℃after centrifuging. Ascites and cyst fluid samples further obtained from five patients were dealed with the same method. No treatment was implemented on all patients before surgery. All of the participants were identified as normal renal function.
All of the tissues matching with the serum were collected at the beginning of the operation and were disposed by paraffin after being fixed with 10% formalin.
2μl Suspention of Weak cation beads (WCX) SPE-CM,95μl of SPE-CB and 100μL of SPE-CW was added into 5μl of serum sample. The prepared samples were obtained after several binging and elution. The ascites and cyst fluid samples were 10μL and 2.5μL respectively for the same preparing method.
1μl of eluent with peptides was mixed with 10μl of matrix (0.3%HCCA) and then 1μl of mixture was applicated onto the Anchorchip. Put the buck sracper into Microflex mass spectrometer (Bruker Daltonics) after calibrated by the standard sample (Clinprot standard). Massspectrograms of peptides peaks with different m/z were obtained after detecting the samples. Then the common elevated peaks in the serum, ascites and cyst fluid were selected for the candidated peptide peaks. At last, the matching peptides were searching in the http://us.expasy.org/tools/tagident. html protein data bank. After retrievaling from prvious data, the deducted cnadidated peptides were selected for next validation.
Immunostaining was performed with the antibodies of the GRP (rabbit polyclonal, MAB35841 R&D Co, Ameraican) and IgG fluorescence Secondary Antibodies (surpported by Yueyan Biotech Co. Shanghai). According to the manufacture's protocol, a standard streptavidin-biotin-peroxidase method was used.
Serum level of GRP was detected by gastrin-releasing peptide ELISA kit (QC01-1, R & D Co, American). The reactions were done by following the manufacture's protocol.
A GRPsiRNA targetting the human GRPmRNA common sequence was constructed and inserted onto the Bam Hi-HindⅡlinearized p-genesil-1 vector. After transfected the plasmid into the ES2 cell, level of GRPmRNA and protein were detected by RT-PCR and Western bloting after the transfection. The proliferation, apoptosis and capability of migration and invasiveness were evaluated by cell dounting, flow cytometry and transwell.
The build-in ClinProtTM software (Bruker Daltonics Company) and FlexAnalysis 3.0 were used to compile the peaks across the spectra and analyze the intensity of single peak. All signals with a signal-to-noise (S/N) ratio>5 in a mass range of 1000-10000 Da were recorded with the ClinProtTM software (Ver.2.2; Bruker Daltonics). A linear SVM classifier was used to distinguish between the different groups of data. The capability of each peak in dstinguishing different groups of data was estimated by the P value of Kruskal-Wallis test. The combinations of the selected peaks was analyzed by the leave-one-out cross-validation. The results of IHC and ELISA were analyzed byχ2 test, q-test and Kruskal-Wallis. Spearman correlations and Pearson correlation coefficient analysis was used to get the relevance of the IHC and ELISA and the GRP and pro-GRP level. Two sample t-test was used to analysize the different level of proliferation, apoptosis, capability of migration, mRNA and protein of GRP before and after transfection in the cell. These data analysis were completed by analysis software SPSS 13.0.
3.Results
MALDI-TOF-MS was used to profile the mass spectral patterns of serum samples from three groups and found that there were no different peaks between benign and normal groups. The common elevated peaks in early and advanced stage of OC were 2881 and 2897Da compared with the normal group (P<0.05). The common elevated peaks in early and advanced stage of OC were 2881,2897 and 4466Da (P<0.05). It was further found that the 2881 and 2897Da peaks were also elevated in the ascite and cyst fluid samples. The pattern of two combined peaks had a specificity of 100% and a sensitivity of 100%, as evaluated by leave-one-out crossvalidation. The remaining 18 serum samples were analyzed as a blind test set. The specificity and sensitivity of the blind test were 100% and 87.5%, respectively.
Compared with normal ovarian group, there were no significant defferential peaks in below and above 50 years old groups (P all>0.05). Compared with normal ovarian group, only 2953 and1466Da peaks were found downregulated in the positive and negative familial history groups (P<0.05) while other differential peaks were thoroughly different.
The candidate peptides matching with 2881 and 2897Da were searching in the http://us.expasy.org/tools/tagident. html peptide data bank. According to the statistic and the reported data, only Gastrin-releasing peptide (GRP) were found closely related to many kinds of cancers among the nine kinds of candidate peptide of OC searching by us.
The positive expression of GRP in tissues of OC were higher than the benign tumor and normal ovary. The expressions of GRP in the normal group were all low to negative expression (χ2= 24.599, P= 0.00). The higher serum level of GRP in OC than benign and normal groups was verified by ELISA (P<0.05, by q-test). There was a high correlation between level of Gastrin-releasing peptide in tissue and serum (r=0.809, P=0.000). The correlations of the serum level of 2881,2897Da peaks and serum level of GRP in three groups were all high. There was an obviously coefficient between GRP and pro-GRP in the training set (r=0.90, P<0.001). Pro-GRP level in serum of training set significantly higher than the benign and normal groups(χ2= 41.81, P=0.00). The area below ROC curve was 0.974 when the proGRP was used for diagnostic marker (z=27.88, P<0.001). The cut-off-value was 89.2851, which had a sensitivity of 100% and a specificity of 90.91%(Youden index was 0.9091) in training set and a sensitivity of 93.33% and a specificity of 80%(Youden index was 0. 7333) in testing set.
Cells stably expressing GRPsiRNA were got by G418 screening after transfection. The proliferation and capability of migration and invasiveness of ES2 cell were significantly decreased (P<0.05) after the transfection accompanied by the down-regulation of the mRNA and protein level of GRP (P<0.05). As a result, the apoptosis of ES2 cell was also activated by the transfection (P<0.05).
4. conclusion
4.1 The 2881 and 2897 Da peaks are highly specific and sensitive serum biomarkers for early detection of OC. The mechanism of the carcinogenesis of hereditary and nonhereditary OC might be partly different
4.2 Gastrin-releasing peptide might be the candidated peptide matching with the 2881 and 2897Da peaks. Pro-GRP had a high diagnostic value in detecting OC with early stage.
4.3. ES2 cell transfected by the GRPsiRNA show a decreased capability of proliferation, migration and invasiveness as well as the activation of the apoptosis. GRP might be a potential target for treatment of OC.
引文
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