膀胱癌组织差异表达蛋白质的筛

发布时间：2018-01-04 03:03

本文关键词：膀胱癌组织差异表达蛋白质的筛选、鉴定及Derlin-1与膀胱癌临床特征的相关性分析　出处：《南京医科大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:膀胱癌是泌尿外科临床上最常见的恶性肿瘤之一。按照膀胱癌恶性程度不同,组织学分级可分为低级别癌和氋级别癌两类;按照其浸润深度可分为非肌层浸润性和肌层浸润性膀胱癌两种。尽管临床上各种手术方式及放化疗手段不断在提高和改进,但膀胱癌治疗后的复发率仍然很高,五年生存率较低,特别是高级别肌层浸润性癌的预后更差。探究膀胱癌的发病机理,寻找有价值的肿瘤标记物和新的治疗靶点,对于指导肿瘤的临床治疗,判断肿瘤的预后具有重要的理论与现实意义。通过研究相关蛋白质在肿瘤中的表达和功能,整体、动态、多角度的研究膀胱癌发生发展过程中的蛋白质组的变化,筛选出与其发生发展相关的特征性蛋白质,可以寻找到具有特异性和敏感性的肿瘤标记物,有助于膀胱癌的早期诊断、治疗以及预后的判断。方法:1.从江苏省人民医院泌尿外科收集144例膀胱癌组织标本。选取3例男性患者的新鲜的高级别浸润性膀胱移行上皮癌组织及癌旁组织,应用Q Exactive质谱仪和生物信息学方法进行差异蛋白分析,初步筛选候选蛋白分子。2.随机选取10对浸润性膀胱癌和癌旁组织,应用qRT-PCR、Western blot分别从mRNA和蛋白水平对候选分子Derlin-1在膀胱癌组织和癌旁组织中的表达差异进行验证。3.采用组织芯片结合免疫组化方法检测Derlin-1蛋白在144例膀胱癌组织和31例癌旁组织中的表达情况;利用卡方检验分析Derlin-1阳性率与膀胱癌临床特征的相关性。采用Kaplan-Meier法进行生存分析,并用Log-rank检验组间差异。用COX比例风险模型进行多因素分析。4.体外细胞实验,首先应用Western Blot技术检测Derlin-1蛋白在人膀胱上皮永生化细胞SV-HUC-1、膀胱癌细胞系UM-UC-3及T24中的表达,然后应用siRNA技术沉默Derlin-1在T24细胞中的表达,应用Transwell培养体系观察转染后的T24细胞迁移和侵袭能力的改变,同时应用Western Blot和免疫荧光技术观察EMT相关蛋白Vimentin、E-cadherin的表达,探讨Derlin-1在膀胱癌中可能的作用机制。结果:1.3例膀胱癌及癌旁组织经质谱分析,共筛选出165种差异蛋白,膀胱癌组织中上调表达19种、下调表达146种。经生物信息学分析这些差异蛋白,其中DERL1(PREDICTED:derlin-1 isoform X1)对应 Derlin-1 蛋白,可能与膀胱癌发生有潜在关联。还筛选出了一些其他的显著差异表达基因ACTA2、ACTN1和VCL,以及焦点粘连通路和ECM受体相互作用等通路。从质谱分析结果中选择膀胱癌相关蛋白Derlin-1为候选蛋白分子。2.qRT-PCR检测结果显示,10例浸润性膀胱癌组织中Derlin-1 mRNA水平是对应的癌旁组织的2.4倍,明显高于癌旁组织,差异有统计学意义(P0.05)。Western blot检测结果显示,浸润性膀胱癌组织中Derlin-1蛋白相对表达量是癌旁组织的3.6倍,是非浸润性膀胱癌组织的1.5倍,差异有统计学意义(P0.05)。3.组织芯片的免疫组化染色显示,Derlin-1在浸润性(98例)和非浸润性(46例)膀肮癌中的阳性率分别为73.5%和47.8%,而癌旁组织(31例)阳性率为32.3%,三者差异有统计学意义(P0.001)。Derlin-1的阳性表达与患者年龄、性别无相关性(P0.05),与肿瘤分期、病理分级、淋巴结转移均呈显著相关性(P0.05)。Kaplan-Meier法分析显示,Derlin-1阳性和阴性表达的膀胱癌患者中位生存期分别为46个月(95%CI:36.3-55.7月)和56个月(95%CI:51.4-60.6月);Derlin-1阳性表达的膀胱癌患者五年生存率明显低于阴性患者,分别为21.79%和46.67%,两者差异有统计学意义(P0.001)。COX多因素分析结果显示,Derlin-1阳性、肿瘤分期以及淋巴结转移是预测膀胱癌独立预后因素(P0.05)。4.在体外细胞实验中,Western Blot技术检测Derlin-1蛋白在人膀胱上皮永生化细胞SV-HUC-1,膀胱癌细胞系UM-UC-3及T24中的表达结果显示,Derlin-1蛋白在UM-UC-3、T24中相对表达量高于在SV-HUC-1中,尤其在T24细胞中表达更高,是UM-UC-3细胞中表达的6.646倍,是SV-HUC-1细胞中表达的20.038倍,三者间差异有统计学意义(P0.01)。T24细胞siRNA沉默Derlin-1基因表达后,Western Blot和免疫荧光结果显示,Derlin-1蛋白表达量下降,与对照组的T24细胞相比,基因沉默后的T24细胞Vimentin蛋白低表达,E-cadherin蛋白高表达,差异有统计学意义(P0.001)。另外,Transwell培养体系结果显示,基因沉默的T24细胞的迁移和侵袭数量低于未转染的T24细胞,差异有统计学意义(P0.05)。结论:1.蛋白质谱分析结合生物信息学分析方法在膀胱癌组织中筛选并鉴定出165种差异表达蛋白质,上调的蛋白有19种,下调的蛋白有146种。其中DERL1(PREDICTED:derlin-1 isoform X1)对应 Derlin-1 蛋白。2.Derlin-1在膀胱癌组织中高表达,与膀胱癌的肿瘤分期、病理组织学分级以及淋巴结转移有关,阳性表达的患者五年生存率明显降低。肿瘤分期、Derlin-1阳性以及淋巴结转移是预测膀胱癌独立预后因素。3.Derlin-1在膀胱癌细胞系中高表达,Derlin-1在膀胱癌发生发展过程中的作用可能与EMT机制有关。Derlin-1有望成为潜在的膀胱癌诊断和预测预后的新的生物学靶点。
[Abstract]:Objective: bladder cancer is one of the most common malignant tumor of the clinical department of urology. According to the different malignant degree of bladder cancer, histological grade can be divided into low grade cancer and Meng grade cancer two; according to the depth of invasion can be divided into non muscle invasive and muscle invasive bladder cancer two. Although all kinds of surgery methods clinical and the chemotherapy means constantly improving and improving, but the treatment of bladder cancer recurrence rate is still high, the five year survival rate is low, especially the high level of muscle invasive cancer prognosis. The pathogenesis study of bladder cancer, tumor marker for valuable and new therapeutic targets for clinical treatment of tumors, it has important theoretical and practical significance in judging the prognosis of tumor. By studying the expression and function of proteins in tumors, the overall, dynamic, the development process of protein in bladder cancer research angle The change group, were correlated with the occurrence and development of characteristic proteins, can find tumor markers with specificity and sensitivity, it is helpful to the early diagnosis of bladder cancer, treatment and prognosis. Methods: 1. from the Department of Urology of Jiangsu Province People's Hospital collected 144 cases of bladder cancer tissue specimens from 3 cases of male patients. Fresh high grade invasive bladder transitional cell carcinoma tissues and adjacent tissues, differences in protein analysis method using Q Exactive mass spectrometry and bioinformatics, preliminary screening of candidate protein.2. randomly selected 10 of invasive bladder cancer and application of qRT-PCR, Western, mRNA and blot respectively from the protein level of the expression of candidate molecules of Derlin-1 in bladder cancer tissues and adjacent tissues of the difference.3. is validated by using tissue chip combined with immunohistochemical detection of Derlin-1 protein in 144 cases of bladder cancer Expression of tissue and 31 cases of adjacent tissues; the correlation analysis of clinical characteristics and the positive rate of Derlin-1 in bladder cancer by chi square test. Survival analysis was performed by Kaplan-Meier method, and Log-rank test the differences between groups..4. analysis of in vitro experiments using COX proportional hazards model for multivariate, the first application of the detection of Derlin-1 protein Western Blot in human bladder cell SV-HUC-1 expression and biochemical Pillon, bladder cancer cell lines UM-UC-3 and T24, and then the application of siRNA technology to silence Derlin-1 expression in T24 cells were observed in T24 system, cell migration and invasion ability changes after dyeing by Transwell culture, at the same time the application of EMT related protein Vimentin Western Blot and immunofluorescence observation technology, the expression of E-cadherin, explore the possible mechanisms of Derlin-1 in bladder carcinoma. Results: 1.3 cases of bladder carcinoma and paracancerous tissues were subjected to mass spectrometry analysis. Identified 165 different proteins in bladder cancer tissues 19 up-regulated, down regulated expression of 146. Bioinformatic analysis of these proteins, including DERL1 (PREDICTED:derlin-1 isoform X1) corresponding to Derlin-1 protein may have potential association with bladder cancer. The expression of ACTA2 gene also screened out some other significant differences, ACTN1 and VCL, as well as the focal adhesion pathway and ECM receptor interaction pathway. Selection of bladder cancer associated protein Derlin-1 from mass spectrometry analysis results show that as a candidate protein molecular testing results of.2.qRT-PCR and Derlin-1 mRNA levels in 10 cases of invasive bladder cancer is 2.4 times higher than paracancerous tissues, adjacent tissues, there were significant differences (P0.05).Western blot assay showed that the invasive bladder cancer tissues the expression of Derlin-1 protein is 3.6 times that of the adjacent tissue, non invasive bladder cancer 1.5 times group, the difference was statistically significant (P0.05) immunohistochemical.3. tissue microarray results in invasive Derlin-1 (98 cases) and non invasive (46 cases) positive rate of bladder cancer were 73.5% and 47.8%, and the adjacent tissues (31 cases) positive rate was 32.3%. There was a significant difference between the three groups (P0.001) positive.Derlin-1 expression and age, no correlation between gender (P0.05), and tumor staging, pathological grading, lymph node metastasis was significantly correlated (P0.05) analysis showed that the.Kaplan-Meier method, the expression of Derlin-1 positive and negative patients with bladder cancer. The median survival time was 46 months (95%CI:36.3-55.7 months) and 56 months (95%CI:51.4-60.6 months); the positive expression of Derlin-1 in bladder cancer patients five years survival rate was significantly lower than negative patients were 21.79% and 46.67%, the difference was statistically significant (P0.001) multivariate.COX analysis showed that Derlin-1 positive , tumor stage and lymph node metastasis is the independent prognostic factor for bladder cancer prediction (P0.05).4. in vitro, detect Derlin-1 protein Western Blot in human bladder on biochemical Pillon cell SV-HUC-1, the expression of bladder cancer cell lines UM-UC-3 and T24 showed that Derlin-1 protein in UM-UC-3, T24 relative expression quantity is higher than that in in SV-HUC-1, especially in T24 cells was 6.646 times higher, the expression of UM-UC-3 in cells, is 20.038 times the expression in SV-HUC-1 cells, there was statistically significant difference between the three (P0.01).T24 expression in siRNA cells after Derlin-1 gene silencing, Western Blot and immunofluorescence analysis showed that Derlin-1 protein expression decreased, compared with and the control group of T24 cells, the low expression of gene silencing in T24 cells after Vimentin protein, E-cadherin protein expression, the difference was statistically significant (P0.001). In addition, the results of Transwell training system Show that the number of migration and invasion of T24 cells in gene silencing than non transfected T24 cells, the difference was statistically significant (P0.05). Conclusion: 1. protein mass spectrometry combined with bioinformatics analysis method in bladder cancer screening and identified 165 differentially expressed proteins, 19 proteins up-regulated, down regulated protein 146. The DERL1 (PREDICTED:derlin-1 isoform X1) corresponding to Derlin-1 high expression of.2.Derlin-1 protein in bladder cancer, and bladder cancer staging, histological grade and lymph node metastasis, the positive expression of the five year survival rate of patients decreased significantly. Derlin-1 positive tumor staging, and lymph node metastasis are independent prognostic factors for predicting.3.Derlin-1 bladder cancer is highly expressed in bladder cancer cell line, Derlin-1 takes effect in the process of the development of EMT and related to the mechanism of.Derlin-1 in bladder cancer is expected to become Potential new biological targets for the diagnosis and prognosis of bladder cancer.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.14

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