【作者】 傅斌；

【导师】 张旭；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 Wnt信号通路广泛地涉及到机体的发育过程与肿瘤的发生。目前有关Wnt信号通路的研究主要集中于Wnt下游信号的异常表现,如β-catenin,APC,GSK-3β,CTNNB1及axin基因突变等。而对于Wnt信号通路的上游信号(Wnt蛋白及其抑制物等)的异常改变与肿瘤发生的关系的研究相对较少。Wnt蛋白的表达在膀胱尿路上皮恶变过程中明显增加:浅表性膀胱肿瘤高于正常膀胱粘膜,而且明显高于侵袭性膀胱癌,这提示Wnt信号通路与膀胱癌发生有关。而Wnt信号通路下游分子,如APC和β-catenin的遗传学改变在膀胱癌中比较少见。因此,我们推测膀胱癌Wnt信号通路上游水平的异常改变可能是激活该通路的主要原因。WIF-1 (Wnt inhibitory factor-1)是Wnt信号通路抑制物之一,它可通过直接与Wnt信号蛋白结合而抑制Wnt信号通路。膀胱癌WIF-1表达下调,但其机制及其生物学效应尚不清楚。一.目的:1.探讨膀胱癌Wnt信号途径上游关键调控分子WIF-1启动子甲基化是否为抑制WIF-1表达的主要原因;研究WIF-1重新表达后对膀胱癌细胞的影响。2.探讨WIF-1的重表达与喜树碱细胞毒性之间的关系,观察膀胱癌WIF-1基因重表达后HCPT抗癌效应的变化,探讨5-aza-dc与HCPT可能协同作用的机制。3.探讨WIF-1启动子甲基化状态与膀胱癌的临床病理关系,进一步明确WIF-1启动子甲基化与膀胱癌的关系,分析WIF-1甲基化可否作为判断膀胱癌患者的预后以及早期诊断的分子标志物。二.方法:1.通过RT-PCR检测Wnt1,Wnt5a,Wnt10b和Wnt13的mRNA在膀胱癌细胞株BIU87和T24的表达情况;通过免疫组织化学技术检测Wnt1在正常膀胱上皮和膀胱癌的表达情况;2.通过RT-PCR、免疫细胞化学以及Western-blot等方法检测膀胱癌细胞株BIU87和T24的WIF-1的表达情况。3.甲基化抑制剂5-aza-dc处理膀胱癌细胞株BIU87和T24,通过甲基化特异性PCR(MSP)方法检测膀胱癌细胞WIF-1基因在5-aza-dc处理前后的甲基化状态的变化;4.5-aza-dc处理膀胱癌细胞BIU87和T24后,通过MTT法,流式细胞仪分析(AnnexinⅤ/PI双染法)和TUNEL法检测膀胱癌细胞的生长抑制及凋亡情况;5.不同浓度的5-aza-dc和羟基喜树碱联合处理膀胱癌细胞株BIU87、T24以及体外原代培养膀胱癌细胞,通过MTT法检测它们的细胞毒性。等效线图解法分析它们的联合效应;6.流式细胞仪分析(AnnexinⅤ/PI双染法)和TUNEL法检测5-aza-dc和羟基喜树碱对膀胱癌细胞的诱导凋亡效应;RT-PCR法检测两药联合作用时对WIF-1表达的影响; 7.MSP法检测膀胱癌组织,正常膀胱组织以及腺性膀胱炎组织标本的WIF-1甲基状态;免疫组织化学技术检测上述组织WIF-1蛋白表达情况。SPSS12.0进行统计分析。三.结果:1. BIU87和T24的Wnt表达情况不尽相同:它们均不表达Wnt1;均表达Wnt13;BIU87不表达Wnt5a,但T24表达; Wnt1在不同分化程度的膀胱癌以及正常膀胱上皮中均不表达;2.正常培养条件下的膀胱癌细胞株BIU87和T24均不表达WIF-1。在5-aza-dc(5μM)作用72小时后,BIU87和T24均表达WIF-1。3. MTT法显示5-aza-dc对膀胱癌细胞有明显的生长抑制作用,与空白对照组比较,差异有显著性意义(p<0.05),两株膀胱癌细胞T24和BIU87之间无显著性差异。5-aza-dc作用72小时后的两种膀胱癌细胞株的凋亡率,与空白对照组比较有显著性差异。4.WIF-1 mRNA的表达不受HCPT的影响,而且HCPT单独作用于膀胱癌细胞并不能恢复WIF-1的表达。5.5-aza-dc和HCPT对膀胱癌细胞有剂量依赖性细胞毒性(p<0.01),两药产生了明显的协同作用;而且2种膀胱癌细胞株与原代培养的3株膀胱癌细胞表现出类似的效果。WIF-1重表达后可以增加HCPT诱导膀胱癌细胞的凋亡。6.WIF-1启动子甲基化阳性率肿瘤分级的增加逐渐升高(G1、G2、G3级分别为20.0%、56.3%和86.7%),组间比较有显著性差异(p<0.05);浅表性和浸润性肿瘤的WIF-1启动子甲基化阳性率也有显著性差异(P<0.05)。通过Kaplan Meier分析显示WIF-1启动子甲基化与未甲基化两组之间的5年总生存率分别为60.6%和91.7%(p<0.05)。7.正常膀胱组织的WIF-1蛋白表达率为92.3%;腺性膀胱炎的WIF-1蛋白表达率为85%,两者之间无明显差异(p>0.05)。膀胱癌组织WIF-1蛋白总体表达率为30.1%,明显低于正常膀胱和腺性膀胱炎的表达率(P<0.01)。WIF-1在浅表性和浸润性肿瘤组织中的表达有显著性差异(p<0.05),而且随肿瘤分级的增加WIF-1阳性表达率逐渐降低,高分化与低分化肿瘤之间差异非常明显(P<0.01)。四.结论:1.膀胱癌WIF-1基因启动子甲基化是抑制WIF-1表达的主要机制;2.WIF-1基因对膀胱癌生物学行为有重要影响,它是膀胱癌的重要抑癌基因。WIF-1启动子去甲基化后恢复表达是5-aza-dc与HCPT协同作用的重要机制;3.WIF-1启动子甲基化与膀胱癌病理分级分期以及生存率密切相关。WIF-1不仅是膀胱肿瘤发生的早期分子事件,而且与膀胱癌的发展有关。WIF-1基因启动子甲基化状态可作为诊断膀胱癌并且判断其预后的一个重要分子标志物。更多还原

【Abstract】 Wnt passway widely involves the development of organisms and tumorigenesis. Aberrant activation of Wnt passway has been found in various cancers including tumors of colon, lung, head and neck, melanoma and leukemia. Present researches commonly were focused on the changes of downstream signals of Wnt passway, such as mutation ofβ-catenin,APC,GSK-3β,CTNNB1, axin and so on . Relatively Fewer studies had been done to illuminate the relation between tumor behaviors and upstream signals including Wnt proteins and their inhibitors. Recent studies showed that colon cancer is related with the abnormal changes of upstream signals of this passway. Wnt protein expression increases in bladder urothelium during tumorigenesis, which means it functions on the development of bladder cancer. Previous studies indicated that the genetic mutations of downstream signal molecules, such as APC andβ-catenin, were less common in bladder cancer. Hence, we hypothesized that abnormity of upstream signals may activate the Wnt passway in bladder cancer. Wnt inhibitory factor-1 can block the signal transduction by direct binding to Wnt protein. Immunohistochemistry revealed the down-regulation of WIF-1 in bladder cancer. However the underlying mechanism of down-regulation of WIF-1 and its relative biological influences on bladder cancer remain unclear.1. Objectives:(1) To detect the expression of Wnt genes in bladder cancer.(2) To test whether promoter methylation of WIF-1 inhibits the gene expression and to study the biological effect of WIF-1’s re-expression on bladder cancer cells.(3) To explore the relation between WIF-1 re-expression and the cytotoxicity of hydroxycamptothecin, and to explore the potential machinery of synergism of 5-aza-2′-deoxycytidine and HCPT by analyzing their combination effect on bladder cancer cells.(4) To explore the relation between WIF-1 promoter methylation and clinical pathology of bladder cancer for the purpose of demonstrating if WIF-1 can be used as a molecular marker for the diagnosis and prognosis prediction of bladder cancer.2. Methods:(1) RT-PCR was used to determine the expression of Wnt1, Wnt5a, Wnt10b and Wnt13 mRNA in bladder cancer cell lines BIU87 and T24; Immunohistochemistry was performed on normal bladder mucosa and bladder cancer tissues to determine the Wnt1 protein expression.(2) RT-PCR, immunocytochemistry and Western-blot analysis were applied to assess the expression pattern of WIF-1 on bladder cancer cell line BIU87 and T24.(3) Demethylating agent 5-aza-2′-dexoycytidine was used to treat bladder cancer cell line BIU87 and T24 in vitro. Methylation-specific polymerase chain reaction (MSP) assay was performed to detect the change of methylation status of WIF-1 gene in bladder cancer cells after the 5-aza-2′-dexoycytidine treatment.(4) Methyl thiazolyl tetrazolium assay (MTT) was used to evaluate the cytotoxicity, and flow cytometry (AnnexinⅤ/PI staining) and terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL) were employed to detect apoptosis of bladder cancer cells after 5-aza-2′-deoxycytidine treatment in vitro.(5) The cytotoxicity of HCPT in combination with 5-aza-dc on bladder cancer cell lines and primary cultured bladder cancer cells was assessed by MTT assay. Isobologram analysis was used to determine if combination effect was synergistic, or additive or antagonistic.(6) FCM and TUNEL were performed to detect the apoptosis of bladder cancer cell lines and primary cultured bladder cancer cells after treatment of HCPT in combination with 5-aza-dc. Then RT-PCR was performed to assess the expression of WIF-1.(7) MSP was applied to evaluate the methylation status of WIF-1 gene of different bladder tissues, including bladder cancer, normal bladder mucosa and glandular cystitis. WIF-1 protein expressions in the tissues were examined by immunohistochemistry. Statistical analysis was performed using SPSS12.0 software.3. Results:(1) Wnt expression pattern of BIU87 and T24 cell was not identical: no Wnt1 expression in both cells, but Wnt13 expressed in both cells; Wnt5a was only seen in BIU87. Wnt1 did not express in bladder cancer tissues and normal bladder mucosa.(2) WIF-1 did not expressed in bladder cancer cell line BIU87 and T24, but re-expressed after 5-aza-dc treatment in both cell lines.(3) 5-aza-dc showed significant effects of growth inhibition and apoptosis-induction on bladder cancer cells compared with control group (p<0.05).(4) HCPT did not interfere with the expression of WIF-1 mRNA when combined with 5-aza-dc in vitro, and HCPT also did not recover the expression of WIF-1.(5) Dose-dependent cytotoxicity of 5-aza-dc and/or HCPT was observed on bladder cancer cells. 5-aza-dc enhanced synergistically the cytotoxicity of HCPT on bladder cancer cell which was related to WIF-1 re-expression.(6) WIF-1 methylation rates increased with the increase of bladder cancer grade (20.0% in G1, 56.3% in G2,and 86.7% G3) and stage (superficial and invasive) (p<0.05). Total 5-year survival of patients with WIF-1 gene promoter methylation was significantly lower that of patients without WIF-1 gene promoter methylation (60.6% versus 91.7%, p<0.05).(7) Positive WIF-1 protein expression did not keep pace with the methylation status of WIF-1, which was lower than the expected percentage. Total expression rate of WIF-1protein expression was observed in 30.1% of patients while 92.3% in normal mucosa and 85% in glandular cystitis. The difference between neoplasia and non-neoplasia was statistically significant (p<0.01).4. Conclusions: (1) DNA methylation was a potential mechanism for silencing the expression of WIF-1 gene in bladder cancer.(2) WIF-1 was an important anti-oncogene for bladder cancer which showed significant inhibitory effect on bladder cancer. WIF-1 re-expression may potentially involve the synergism of 5-aza-dc and HCPT on bladder cancer cells.(3) The methylation status of WIF-1 gene was significantly associated with bladder cancer grade and stage, and long-term survival of patients. Therefore WIF-1 gene promoter methylation can be used as a good molecular marker for the diagnosis and prognosis prediction for bladder cancer.更多还原