钙黏附蛋白CDH11在膀胱癌中生物学功能及其临床意义

发布时间：2018-09-18 10:17

【摘要】：背景和意义膀胱癌是全世界常见恶性肿瘤之一,居全球恶性肿瘤发病率第11位,约占全球肿瘤发病率的3%。2012年全球新增膀胱癌429800例,165100例患者死于膀胱癌。膀胱癌好发于男性,男性发病率大概是女性的3-4倍。膀胱癌发病率较高的是欧洲、北美、西亚以及东非,中非、西非以及东方国家及地区的发病率相对较低。在美国,膀胱癌居男性恶性肿瘤发病率的第4位,据预测2015年美国将会新诊断74000例膀胱癌患者,而且将会有16000名患者因膀胱癌而死亡。全球男性膀胱癌死亡率最高的是土耳其,据估算,土耳其男性膀胱癌的死亡率(12.8/10万)是美国的3倍,同时比欧洲最高死亡率的国家拉脱维亚(8.3/10万)高出50%。在中国泌尿外科,膀胱癌是发病率最高的恶性肿瘤,2012年中国膀胱癌发病率在男性恶性肿瘤中居第6位,新增男性膀胱癌病人5.7万。1998至2008年中国膀胱癌发病率呈逐年增长趋势,膀胱癌占中国肿瘤发病的构成比例由1998年的2.21%增高至2008年的2.5%,居中国恶性肿瘤发病率第8位,占中国恶性肿瘤发病构成的2.5%。膀胱癌分为非肌层浸润性膀胱癌(NMIBC)和肌层浸润性膀胱癌(MIBC)。据统计,初诊时70%-80%膀胱癌为分化良好或中等分化的NMIBC,首次经尿道膀胱肿瘤切除(TURBT)术后,NMIBC第一年复发的几率为15%-61%,五年内复发率为31%-78%。其中约10%的患者最终进展为肌层浸润性膀胱癌或者转移性膀胱癌；尤其是高危非肌层浸润膀胱癌,行经尿道膀胱肿瘤切除术后5年进展的几率达45%。侵袭和转移是膀胱癌患者的首要死因,国内一项研究显示,各期膀胱癌患者5年生存率分别为：Ta-1期膀胱癌91.9%,T2期84.3%、T3期43.9%、T4期10.2%。国外研究报道,对于肿瘤局限膀胱内并且无淋巴结转移的非肌层浸润性膀胱癌5年和10年无复发生存比例分别为：P0期92%和86%,Pis期91%和89%,Pa期79%和74%,P1期83%和78%。对于肌层浸润性膀胱癌,若淋巴结阴性,5年无复发生存率和10年无复发生存率分别为：T2期：89%和87%,T3a期：78%和76%,T3b期：62%和61%,T4期：50%和45%。而淋巴结阳性患者的5年和10年无复发生存率只有35%和34%。由于膀胱癌患者的生物行为存在明显异质性,所以现在治疗指南推荐紧密的随访和有创的治疗,造成患者大量的经济负担。因此,准确判断出哪些肿瘤具有复发和进展的潜能极其临床意义。有一部分学者认为对于反复复发和容易进展的高危非肌层浸润性膀胱癌应该尽早行根治性全膀胱切除以达到最好的肿瘤控制效果[9]。病理的分级、分期等曾是判断肿瘤预后最好的临床指标之一,例如EORCT评分系统,将其作为判断术后NMIBC复发和进展的几率的重要风险因子；但是在指导个体化治疗方面,其精确度欠佳。目前,在寻找预测膀胱癌进展和复发的生物标记物方面做了大量工作,但是仍然没有较为理想的生物标记物应用临床实践中。据此,本项研究拟利用GEO数据库下载的膀胱癌基因表达谱进行深度挖掘,发现NMIBC和MIBC的差异表达基因,并且通过大样本回顾性分析进行验证,探讨影响NMIBC术后复发进展的关键调节基因。在初步研究中我们发现CDH11是促进NMIBC进展的关键因子之一,并初步探讨其在膀胱癌的生物学功能,在临床样本中验证生物学信息的准确性,并阐明其临床意义。对深入研究NMIBC进展的分子机制、指导NMIBC的精准治疗和改善NMIBC的预后具有重要的理论和临床意义。材料和方法1.研究对象1.1膀胱癌组织标本所有标本收集于2007年1月-2014年5月,来自南方医科大学第三附属医院和中山大学附属肿瘤医院泌尿外科。标本分为三组：1.用于实时荧光定量PCR实验的59例冰冻膀胱癌组织和21例癌旁正常膀胱组织；2.用于Western Blot实验的10例冰冻膀胱癌组织和4例癌旁组织。3.用于免疫组化实验的209例石蜡膀胱癌组织以及12例癌旁组织。所有样本经南方医科大学第三附属医院及中山大学附属肿瘤医院的病理科专家明确病理诊断。1.2膀胱癌基因表达谱数据从美国生物技术信息GEO数据库(http://www.ncbi.nlm.nih.gov/gds/)下载4套基因表达谱,登录号分别为GSE3167 GSE37317,GSE31684和GSE5287,筛选后将132例肌层浸润性膀胱癌和56例非肌层浸润性膀胱癌组织的基因芯片数据纳入实验分析。2.研究方法2.1基因芯片数据分析使用R语言软件,采用RMA方法和用Entrez基因为中心的CDF文件代替原始的Affymetrix的CDF文件重新归纳基因表达谱数据,以过滤GeneChips上的非特异探针,并且将代表同一Entrez基因的不同探针合并在一个探针集中。采用SAM方法分析MIBC和NMIBC组织之间的差异表达基因,筛选差异表达基因的标准：A=2.25,FDR=0.001。随后采取GenCLiP软件(网站：http://ci. smu.edu.cn)对差异表达基因进行后续的Pathway和GO功能注释。2.2实时荧光定量PCR分析采用实时荧光定量PCR方法检测59例膀胱癌组织以及21例癌旁组织中CDH11mRNA的表达量。TBP作为内参基因,采用2-△△Ct相对定量法来分析和反映样本间CDH11基因的表达差异。2.3 Western Blot分析随机挑选5例NMIBC、5例MIBC膀胱癌标本和4例癌旁标本,提取总蛋白进行Western Blot实验,用GAPDH作为内参,进行半定量分析。2.4免疫组化分析收集了209例膀胱癌石蜡组织(包括22例MIBC和187例NMIBC)以及12例正常膀胱组织。使用SP法免疫组化实验检测CDH11蛋白在膀胱癌组织和正常膀胱组织中的表达情况。免疫组化实验评分判断标准：每张玻片在高倍镜下观察5个视野。染色强度得分为：无(0分)、轻度(1分)、中度(2分)和重度(3分)；阳性表达细胞占总肿瘤细胞的比例得分为：无(0分)、≤25%(1分)、≤50%(2分)、≤75%(3分)和75%(4分)。总得分=染色强度得分+阳性表达细胞占总肿瘤细胞的比例得分,总分范围0-7分。阳性定义：只要有细胞膜阳性着色(总得分≥1)；阴性定义：细胞膜完全没有阳性着色(总得分=0)。2.5Transwell和Boyden实验使用Transwell和Boyden实验检测CDH11对膀胱癌细胞株T24、BIU87、EJ和5637迁移侵袭能力的影响。2.6统计分析采用SPSS 20.0统计软件分析实验数据。除特别说明外,采用t检验统计分析两组间数据；使用卡方检验分析CDH11与临床指标的关系,使用Kaplan-Meier and log-rank tests方法进行生存分析以及单因数分析,多因素分析使用COX风险模型。P0.05有统计学意义。结果1.生物信息学分析结果提示：CDH11等基因介导的EMT现象与NMIBC术后进展密切相关基因表达谱分析结果显示：414个基因存在差异表达,其中在肌层浸润性膀胱癌中表达上调的基因有185个,表达下调的基因有229个。使用genclip软件对差异表达基因进行基因功能注解,GO分析发现这些差异表达基因与细胞粘附、细胞运动、细胞增殖、细胞凋亡等有关,pathway分析发现差异表达基因主要与整合素信号通路、TGF-β信号通路和细胞外基质等信号通路相关。此外,在414个差异表达基因中有164个基因与EMT相关；其中CDH11基因表达水平差异表达较大,达2.89倍,P0.0001。2.CDH11mRNA及蛋白在膀胱癌组织中表达上调实时荧光定量PCR和Western Blot实验分别检测CDH11 mRNA和蛋白在膀胱癌组织中的表达水平。发现相对膀胱正常组织,膀胱癌中CDH11的mRNA和蛋白表达水平均升高,而且MIBC组织中CDH11的表达量高于NMIBC,差异具有统计学意义。免疫组化实验结果显示：正常膀胱上皮组织不表达CDH11,CDH11表达于肿瘤细胞的细胞膜。NMIBC中CDH11表达阳性率为30.5%(57/187),而MIBC中CDH11表达阳性率为54.5%(12/22),差异表达具有统计学意义。3.CDH11促进膀胱癌细胞迁移和侵袭。实时荧光定量PCR和Western Blot分析发现T24和BIU87细胞株中CDH11mRNA和蛋白表达水平相对较高,而5637和EJ细胞株中表达相对较低;Transwell和Boyden实验发现CDH11促进膀胱癌细胞的迁移和侵袭能力。4.CDH11在膀胱癌中的临床意义临床病理资料分析发现CDH11阳性表达与膀胱癌的肿瘤分级、分期、复发、进展等临床指标呈正性相关;阳性表达CDH11的患者更容易复发和进展。单因素分析和多因素分析发现CDH11阳性表达是影响NMIBC患者的无复发和无进展生存时间的独立危险因素。结论：1.通过基因芯片以及生物信息学技术筛选出与膀胱癌进展相关的大量差异表达基因,分析发现这些基因主要涉及整合素信号通路、TGF-P信号通路和细胞外基质等信号通路；且与细胞粘附、细胞运动、细胞增殖、细胞凋亡等有关；而且这些差异表达基因主要与EMT现象相关。2.CDH11 mRNA和蛋白在膀胱癌组织中表达上调,而且肌层浸润性膀胱癌中的表达水平高于非肌层浸润性膀胱癌。3.CDH11在T24和BIU87细胞株中相对高表达,在EJ和5637细胞株中相对低表达,具有促进膀胱癌细胞迁移和侵袭的能力。4.CDH11与膀胱癌分级、分期、复发、进展等临床指标呈正相关,CDH11阳性表达提示患者预后不良。CDH11也许可以作为预测膀胱癌预后的生物标记物。
[Abstract]:BACKGROUND AND SIGNIFICANCE Bladder cancer is one of the most common malignant tumors in the world, ranking 11th in the incidence of malignant tumors worldwide, accounting for about 3% of the global cancer incidence. In 2012, 429 800 new bladder cancer cases, 165,100 patients died of bladder cancer. Bladder cancer is predominant in men, and the incidence of male is about 3-4 times higher than that of female. Bladder cancer is the fourth most common malignancy in men in the United States. It is estimated that 74,000 new bladder cancer cases will be diagnosed in the United States in 2015 and 16,000 deaths from bladder cancer will occur worldwide. Turkey has the highest rate of bladder cancer. It is estimated that the mortality rate of male bladder cancer in Turkey (128/100,000) is three times higher than that in the United States and 50% higher than that in Latvia (8.3/100,000), the European country with the highest mortality rate. From 1998 to 2008, the incidence of bladder cancer in China increased from 2.21% in 1998 to 2.5% in 2008, ranking eighth in the incidence of malignant tumors in China, accounting for 2.5% in the incidence of malignant tumors in China. Bladder cancer (NMIBC) and myometrial invasive bladder cancer (MIBC). According to statistics, 70% to 80% of bladder cancer at first diagnosis is well-differentiated or moderately differentiated NMIBC. After the first transurethral resection of bladder tumor (TURBT), the recurrence rate of NMIBC in the first year is 15% - 61%, and the recurrence rate is 31% - 78% within five years. Invasion and metastasis are the leading causes of death in patients with bladder cancer. According to a domestic study, the 5-year survival rates of patients with various stages of bladder cancer are 91.9% for Ta-1, 84.3% for T2, 43.9% for T3, and 43.9% for T4, respectively. The 5-year and 10-year survival rates of non-myometrial invasive bladder cancer with localized bladder tumor and no lymph node metastasis were 92% and 86%, 91% and 89% in Pis stage, 79% and 74% in Pa stage, 83% and 78% in P1 stage, respectively. Recurrence survival rates were 89% and 87% in stage T2, 78% and 76% in stage T3a, 62% and 61% in stage T3b, 50% and 45% in stage T4, respectively. In lymph node-positive patients, 5-year and 10-year recurrence-free survival rates were only 35% and 34% respectively. Some scholars believe that radical cystectomy should be performed as soon as possible for high-risk non-myometrial invasive bladder cancer with recurrence and easy progression to achieve the best tumor control effect [9]. The EORCT scoring system, for example, has been used as an important risk factor for the recurrence and progression of NMIBC after surgery, but its accuracy in guiding individualized treatment is poor. A great deal of work has been done, but there is still no ideal clinical application of biomarkers. Therefore, this study intends to use the gene expression profiles downloaded from GEO database to dig in depth and find the differentially expressed genes between NMIBC and MIBC, and verify them by a large sample retrospective analysis to explore the impact of NMIBC recurrence after surgery. In the preliminary study, we found that CDH11 is one of the key factors to promote the progress of NMIBC, and preliminarily explore its biological function in bladder cancer, verify the accuracy of biological information in clinical samples, and clarify its clinical significance. Materials and Methods 1.1 All specimens of bladder cancer tissue were collected from January 2007 to May 2014 from the Third Affiliated Hospital of Southern Medical University and the Urology Department of the Cancer Hospital of Sun Yat-sen University. The specimens were divided into three groups: 1. Real-time fluorescence quantitative PCR 59 cases of frozen bladder cancer tissue and 21 cases of normal bladder tissue adjacent to cancer; 2. 10 cases of frozen bladder cancer tissue and 4 cases of adjacent bladder tissue used in the Western Blot experiment. 3. 209 cases of paraffin bladder cancer tissue and 12 cases of adjacent tissues used in immunohistochemistry. All samples were obtained from the Third Affiliated Hospital of Southern Medical University and the Oncologist Affiliated to Zhongshan University. Pathological specialists in the hospital confirmed the pathological diagnosis. 1.2 Bladder oncogene expression profiles were downloaded from the US Biotechnology Information GEO database (http://www.ncbi.nlm.nih.gov/gds/) and registered at GSE3167 GSE37317, GSE31684 and GSE5287, respectively. After screening, 132 cases of myometrial invasive bladder cancer and 56 cases of non-myometrial invasive bladder cancer were selected. Methods 2.1 Gene chip data analysis used R language software, RMA method and CFD file with Entrez gene as the center were used to replace the original CFD file of Affymetrix to re-summarize gene expression profile data to filter non-specific probes on GeneChips and represent the same. Different probes of Entrez gene were merged into one probe set. SAM method was used to analyze the differentially expressed genes between MIBC and NMIBC tissues, and the criteria of differentially expressed genes were screened: A = 2.25, FDR = 0.001. GenCLiP software (website: http://ci.smu.edu.cn) was then used to annotate the differentially expressed genes in Pathway and GO function.2.2. The expression of CDH11 mRNA in 59 bladder cancer tissues and 21 adjacent bladder cancer tissues was detected by real-time fluorescence quantitative PCR. TBP was used as an internal reference gene. The expression of CDH11 gene was analyzed and reflected by 2-delta CT relative quantitative method. 2.3 Western Blot analysis randomly selected 5 cases of NMIBC and 5 cases of MIBC bladder cancer. Total protein was extracted from specimens and 4 adjacent specimens for Western Blot assay. GAPDH was used as internal reference for semi-quantitative analysis. 2.4 Immunohistochemistry was used to collect paraffin tissues from 209 cases of bladder cancer (including 22 cases of MIBC and 187 cases of NMIBC) and 12 cases of normal bladder tissues. Normal expression of bladder tissue. Immunohistochemical test score criteria: each slide was observed under high power microscope 5 visual fields. The staining intensity scores were: no (0 points), mild (1 point), moderate (2 points) and severe (3 points); the proportion of positive cells in total tumor cells was: no (0 points), < 25% (1 point), < 50% (2 points), < 75% (3 points). Positive definitions: as long as there is positive staining of the cell membrane (total score (> 1); negative definitions: there is no positive staining of the cell membrane (total score = 0). 2.5 Transwell and Boyden experiments used Transwell and Boyden tests. The effects of CDH11 on the migration and invasion of bladder cancer cell lines T24, BIU87, EJ and 5637 were measured. 2.6 The experimental data were analyzed by SPSS 20.0 statistical software. Survival analysis and single factor analysis showed that COX risk model was used for multivariate analysis. P 0.05 had statistical significance. Results 1. Bioinformatics analysis showed that CDH11 and other gene-mediated EMT phenomena were closely related to the progress of NMIBC after operation. Gene expression profiles showed that 414 genes were differentially expressed, including infiltration in muscular layer. There were 185 up-regulated genes and 229 down-regulated genes in bladder cancer. Genclip software was used to annotate the function of differentially expressed genes. GO analysis showed that these differentially expressed genes were related to cell adhesion, cell movement, cell proliferation and cell apoptosis. Path analysis showed that differentially expressed genes were mainly related to integrin signaling. In addition, 164 of 414 differentially expressed genes were associated with EMT, and the expression level of CDH11 gene was 2.89 times, P 0.0001.2. The expression of CDH11 mRNA and protein was up-regulated by real-time quantitative PCR and Western Blot assay in bladder cancer tissues. The expression levels of CDH11 mRNA and protein in bladder cancer tissues were detected respectively. It was found that the expression levels of CDH11 mRNA and protein in bladder cancer tissues were higher than those in normal bladder tissues, and the expression level of CDH11 in MIBC tissues was higher than that in NMIBC tissues. The positive rate of CDH11 expression in NMIBC was 30.5% (57/187), while that in MIBC was 54.5% (12/22). The difference was statistically significant. 3. CDH11 promoted the migration and invasion of bladder cancer cells. Real-time fluorescence quantitative PCR and Western Blot analysis showed that CDH11 mRNA and eggs were found in T24 and BIU87 cell lines. The expression level of CDH11 in bladder cancer was relatively high, while that in 5637 and EJ cells was relatively low. Transwell and Boyden experiments showed that CDH11 promoted the migration and invasion of bladder cancer cells. The positive expression of CDH11 was found to be an independent risk factor for recurrence-free and progression-free survival in patients with NMIBC by univariate and multivariate analysis. Differentially expressed genes were mainly involved in integrin signaling pathway, TGF-P signaling pathway and extracellular matrix signaling pathway, and were related to cell adhesion, cell movement, cell proliferation, cell apoptosis, etc. These differentially expressed genes were mainly related to EMT phenomena. 2. CDH11 mRNA and protein in bladder cancer tissue surface. CDH11 was relatively high in T24 and BIU87 cell lines, but relatively low in EJ and 5637 cell lines. It has the ability to promote the migration and invasion of bladder cancer cells. 4. CDH11 and bladder cancer classification, stage, recurrence, progression and other clinical indicators were presented. CDH11 may be a biomarker for predicting the prognosis of bladder cancer.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.14

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