膀胱癌CYR61基因异常表达的临床生物学意义及其分子机制

发布时间：2018-05-20 08:00

本文选题：膀胱癌 + 肌层浸润　；参考：《北京协和医学院》2015年博士论文

【摘要】：非肌层浸润性膀胱癌和肌层浸润性膀胱癌的生物学行为、治疗方式和预后均有明显差异。在手术前准确判断肿瘤是否发生肌层浸润,对膀胱癌的治疗方案选择以及预后判断至关重要。目前,临床术前判断膀胱癌是否发生肌层浸润主要依靠膀胱镜和影像学(CT或MRI)检查,但这些方法均有一定的局限性,尚不能充分满足临床实际需求。寻找合适的肿瘤分子标志物对膀胱癌进行分子分期已经成为辅助膀胱癌术前诊断的研究热点。本课题第一部工作在前期采用基因芯片技术获得的非肌层浸润性(16例)和肌层浸润性膀胱癌(14例)mRNA表达谱的基础上,分析两型膀胱癌之间的差异表达基因。首先,通过数据挖掘初筛获得11个显著差异表达基因(AK098422、APOLD1、 C13orf33、CYR61、HSD17B3、LIG4、NR4A2、PCK2、PLK3、RBMS3、RTN4);随后,以实时定量PCR (Realtime PCR)技术,对初筛所得基因在完成上述芯片分析的30例“起始样本”和另一组膀胱癌(54例)“独立样本”中依次进行了验证；最终有4个基因(AK098422、C13orf33、CYR61、RBMS3)在两型膀胱癌中仍显著差异表达。继而,针对其中差异表达最明显的基因CYR61,在115例膀胱癌福尔马林固定石蜡包埋的肿瘤组织中进行了免疫组化染色,发现浸润性癌中的CYR61蛋白强阳性率显著高于非浸润性癌(p=0.001)；然而肿瘤组织中CYR61蛋白表达水平对于患者的无瘤生存期并无明显影响(p=0.571)。鉴于CYR61是典型的分泌蛋白,进而以酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)测定104例膀胱癌患者术前尿中的CYR61蛋白。结果提示,浸润性膀胱癌患者术前尿上清中的CYR61含量显著高于非浸润性癌患者,其鉴别诊断的敏感度为72.7%,特异度为86.0%,诊断效能优于MMP2、VEGF、VEGFC这3个已有报道的相关蛋白标志物；CYR61与此3个蛋白联合鉴别肌层浸润性膀胱癌的敏感性为87.9%,特异度为86.4%。此外,术前尿中CYR61蛋白水平对于膀胱癌肌层浸润的诊断效能亦优于影像学检查CT或MRI。本课题第二部分工作利用体外实验针对CYR61异常表达在膀胱癌侵袭过程中的作用及其表达增高的分子机制进行了探索。首先分析了不同膀胱癌细胞系中内源性CYR61的表达情况,证实浸润性癌细胞系的CYR61基因表达量高于非浸润性癌细胞系。随后的实验结果显示,外源性过表达CYR61基因可以促进浸润性膀胱癌细胞的侵袭和迁移能力；反之,沉默CYR61表达可以抑制浸润性膀胱癌细胞的侵袭和迁移能力；此外,CYR61表达变化可以调节浸润性膀胱癌细胞中MMP2、NRP1的表达。但是,在非浸润性膀胱癌细胞中CYR61的表达变化却并不影响细胞的侵袭和迁移能力,也不能调节非浸润性膀胱癌细胞中MMP2、NRP1的表达。基于前期工作的arrayCG H芯片分析数据,比较了非浸润性膀胱癌(29例)与浸润性膀胱癌(36例)中CYR61基因拷贝数,结果提示DNA拷贝数改变不是CYR61表达升高的原因。结合本实验室基于前期工作的microRNA表达谱芯片分析数据,筛选获得非浸润性和浸润性膀胱癌组织之间差异表达且与CYR61基因有关的3个miRNA,利用荧光双报告实验确定miR-30e可以结合CYR61基因3’UTR区域,调控膀胱癌细胞中CYR61的表达。进而以Realtime PCR检测了72例膀胱癌组织中miR-30e的表达,发现浸润性膀胱癌表达量低于非浸润性癌；而且肿瘤组织中的CYR61与miR-30e的表达量呈负相关；从而提示,miRNA-30e低表达可能是其靶基因CYR61在肌层浸润性膀胱癌中表达升高的原因。本项研究针对前期利用高通量技术分析数据获得的非肌层浸润性和肌层浸润性膀胱癌之间的差异表达基因进行了两轮验证,以及相应的生物学功能探索。课题工作的主要新发现为,揭示了CYR61基因在膀胱癌组织中异常表达的临床意义,及其对于肿瘤浸润过程的作用；明确了尿上清液中CYR61蛋白术前辅助鉴别肌层浸润性膀胱癌的诊断价值,为CYR61作为新型肿瘤分子标志物的临床转化应用研究奠定了基础。
[Abstract]:The biological behavior, treatment and prognosis of non myometrium invasive bladder cancer and myometrium invasive bladder cancer are significantly different. It is crucial to determine whether the tumor is myometrium infiltration before operation and the choice of treatment options and prognosis of bladder cancer. Cystoscopy and imaging (CT or MRI) examination, but these methods have some limitations, can not fully meet the needs of clinical practice. Finding the appropriate molecular markers for the cancer of the bladder has become a hot spot for the diagnosis of bladder cancer. The first work of this topic is to use gene chip technology in the early stage. On the basis of the mRNA expression profiles of non myometrium infiltration (16 cases) and myometrium infiltrating bladder cancer (14 cases), the differential expression genes between type two bladder cancer were analyzed. First, 11 significant differentially expressed genes were obtained by data mining screening (AK098422, APOLD1, C13orf33, CYR61, HSD17B3, LIG4, NR4A2, PCK2, PLK3, RBMS3, RTN4); and then, in real time Quantitative PCR (Realtime PCR) technique was used to verify the initial screening genes in 30 "initial samples" and another group of bladder cancer (54 cases) "independent samples", and 4 genes (AK098422, C13orf33, CYR61, RBMS3) were still significantly different in type two bladder cancer. The most obvious gene CYR61 was immunohistochemical staining in 115 cases of formalin fixed paraffin embedded tumor tissues of bladder cancer. The strong positive rate of CYR61 protein in invasive cancer was significantly higher than that of non invasive cancer (p=0.001). However, the level of CYR61 protein expression in tumor tissues had no significant influence on the tumor free survival of the patients. (p=0.571). Given that CYR61 is a typical secretory protein, and then enzyme-linked immunosorbent assay (ELISA) is used to determine the CYR61 protein in the urine of 104 patients with bladder cancer. The results suggest that the level of CYR61 in the urine supernatant of the patients with invasive bladder cancer is significantly higher than that of non invasive cancer patients, and the sensitivity of the differential diagnosis is sensitive. The degree is 72.7% and the specificity is 86%. The diagnostic efficiency is superior to MMP2, VEGF, VEGFC, the 3 reported related protein markers. The sensitivity of CYR61 and the 3 proteins to identify the invasive bladder cancer is 87.9% and the specificity is 86.4%.. The diagnostic efficiency of the CYR61 protein level in the urine for bladder cancer is also superior to that of the image. The second part of the study, CT or MRI., explored the role of abnormal expression of CYR61 in the invasion of bladder cancer and the molecular mechanism of high expression in vitro. Firstly, the expression of endogenous CYR61 in different bladder cancer cell lines was analyzed, and the CYR61 gene expression of the dipped cancer cell line was proved to be higher than that of the human bladder cancer cell line. The subsequent experimental results showed that exogenous overexpression of CYR61 gene could promote the invasion and migration of invasive bladder cancer cells, and conversely, silent CYR61 expression could inhibit the invasion and migration of invasive bladder cancer cells; in addition, the changes in CYR61 can regulate the MMP2 in invasive bladder cancer cells. The expression of NRP1, however, changes in the expression of CYR61 in non invasive bladder cancer cells does not affect the invasion and migration of cells, nor does it regulate the expression of MMP2 and NRP1 in non invasive bladder cancer cells. Based on the arrayCG H chip analysis of earlier work, non invasive bladder cancer (29 cases) and invasive bladder cancer (36 cases) are compared. The copy number of the CYR61 gene indicates that the change of the DNA copy number is not the cause of the increase in the expression of CYR61. Combined with the microRNA expression chip analysis data based on previous work in our laboratory, the differential expression of non invasive and invasive bladder cancer tissues and the 3 miRNA related to the CYR61 gene are screened and the fluorescence double report test is used. MiR-30e can regulate the expression of CYR61 in bladder cancer cells by combining the CYR61 gene 3 'UTR region. Then, the expression of miR-30e in 72 bladder cancer tissues is detected by Realtime PCR, and the expression of the invasive bladder cancer is lower than that of non invasive carcinoma; and the CYR61 in the tumor tissue is negatively correlated with the expression of miR-30e; thus, miRNA-3 is suggested, miRNA-3. The low expression of 0e may be the cause of the increase in the expression of the target gene CYR61 in myometrium infiltrating bladder cancer. This study has conducted two rounds of verification for the differential expression genes between non muscularis infiltrative and myometrium infiltrating bladder cancer, which were obtained by high throughput technical analysis data, and the corresponding biological function exploration. The main new discovery is to reveal the clinical significance of the abnormal expression of CYR61 gene in bladder cancer and its role in the process of tumor invasion, and to clarify the diagnostic value of CYR61 protein in the urine supernatant to assist in the diagnosis of invasive bladder cancer before operation, and to lay a foundation for the clinical transformation and application of CYR61 as a new tumor molecular marker. Set the foundation.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.14

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