膀胱癌中SphK1表达与疾病预后的研究

发布时间：2018-05-22 12:35

本文选题：膀胱癌 + 鞘氨醇激酶1　；参考：《第三军医大学》2015年博士论文

【摘要】：背景膀胱癌(Bladder cancer,BC)是泌尿系统中易发的首位肿瘤,具有较高的发病率和致死率。目前治疗措施主要有腔内膀胱肿瘤切除,根治性膀胱切除等手术、适形放疗和局部灌注或全身化疗等,但是经上述种种措施单独或合并治疗后复查时还经常出现肿瘤再发甚至转移,预后不良。临床数据分析显示自2007年至2012年共有来自我们国家44个大型泌尿外科中心共14260例膀胱癌患者,其中约70%以上的患者接收治疗后出现再发转移扩散,30%的再发肿瘤恶性度较前增加。随着分子靶向治疗在肿瘤综合治疗中逐渐成为临床热点,膀胱癌中有无合适的分子靶点以及如何利用该靶点来减缓肿瘤的再发和进展将具有肯定的现实性意义。脂类组学的研究发现鞘脂也可以具有信号调控的作用。如神经酰胺(Ceramide,Cer)、鞘氨醇(Sphingosine,Sph),可以调控细胞内信号通路引起细胞周期阻滞和促进细胞凋亡;鞘氨醇1-磷酸(Sphingosine 1-phosphate,S1P)则有促进细胞增殖的功能。Cer/Sph与S1P在细胞中的水平存在动态平衡,而它们的作用一种促死与一种促生,正好相反,水平的平衡决定了作用的平衡,作用的平衡决定了该细胞的命运是活或死。这种现象就是所谓的“鞘脂可调变阻器”(Sphingolipid-rheostat function)。研究认为鞘氨醇激酶族(Sphingosine kinases,SphKs)是调控这个平衡的最重要酶族,它能将促死的鞘氨醇、神经酰胺转化为促生的磷酸鞘氨醇。通过抑制此族蛋白的活性则能将可调变阻器调向细胞的死亡方向起到抗肿瘤的作用。发现Sph K1可作为治疗肿瘤的手段。肿瘤细胞侵袭与转移是影响肿瘤患者预后的重要原因。膀胱尿路上皮癌细胞的转移需要历经癌细胞-基底细胞黏附、细胞-基质黏附的破坏,细胞外介质蛋白的水解,以及新生血管生成的一系列过程,抗肿瘤研究就是要研究如何阻断这一系列过程,进而阻断癌细胞的转移,侵袭及扩散,同样是膀胱癌防治工作的重点。我们已知癌细胞侵袭、迁移的过程中,基质金属蛋白酶家族(Matrix-metalloproteinase,MMPs)的降解既是肿瘤侵袭与癌细胞迁移中组织重构所必需的,也是肿瘤侵袭早期的变化之一。恶变细胞通过游离的MMPs发挥降解细胞外基质、促新生血管形成的双重作用而利于其侵袭。基质金属蛋白酶家族中的成员MMP-2与MMP-9能够降解Ⅳ型胶原蛋白进而促进基质膜的降解。在多种肿瘤细胞的侵袭与转移过程中它们的表达都出现升高,两种现象具有相关性。细胞外反应激酶(extracellularresponsekinases,erk)是mapk蛋白家族中的一种蛋白,有酶活性,其生物学作用是能够控制细胞增殖和分化、调控细胞凋亡、决定细胞恶性变,并且能够维持细胞形态。erk的表达升高及激活状态上调可以在许多的人类恶性肿瘤中检测到。p-erk1/2是erk1/2的活化形式,通过erk1/2的202位l-苏氨酸(l-threonine,thr202)与204位酪氨酸(tyrosine,tyr204)出现磷酸化具有活性,作用较erk1/2增强。文献复习发现在许多肿瘤中sphk1/s1p激活erk1/2通路从而促进癌细胞侵袭与迁移。尿激酶型纤维蛋白溶酶原激活物(urokinase-typeplasminogenactivator,upa)能够将血浆纤维蛋白溶酶原活化为酶并能够独立地或者通过活化的酶降解细胞外基质类物质、激活mmps、引起促肿瘤因子以及vegf的释放促进细胞侵袭性增强,并能加速肿瘤细胞的迁移能力。既往研究发现通过抑制erk1/2通路,经过一系列信号转导、放大、反馈等方式,可出现mmp-2/9、upa表达的下调,它们协同起来可以实现抑制肿瘤转移的目的。本研究即以sphk1作为对象,明确sphk1在bc患者中表达是否存在异常,其水平与临床病理特点及疾病预后是否关联,并将探讨其细胞水平及分子水平的机制。具体方式为应用调控sphk1蛋白表达的化学药物作用于膀胱癌肿瘤细胞biu-87,观察膀胱肿瘤细胞biu-87被药物处理后的侵袭和迁移能力的变化,并测定细胞内erk1/2与perk1/2蛋白,mmp-2、mmp-9、upa及vegf蛋白分泌的变化,从细胞及分子水平明确鞘氨醇激酶1有无改变人膀胱癌细胞的侵袭和迁移能力的作用,其作用是增强还是减弱,进而与临床病理结果相对应,为以鞘氨醇激酶1为靶点的分子肿瘤治疗学初步言明机制。研究目的探讨sphk1在膀胱尿路上皮癌发生发展中的作用及其机制,为将sphk1作为膀胱尿路上皮癌的诊断治疗之分子靶点提供理论依据。材料、方法和结果以细胞及其中分子为研究对象用佛波醇12-肉豆蔻酸酯13-乙酸酯(phorbol12-myristate13-acetate,pma)为sphk1激活剂;二甲基鞘氨醇(n-dimethyld-erythrosphingsine,dms)为sphk1抑制剂;0.9%nacl为空白试剂将膀胱癌biu-87细胞株分别处理为激活组、抑制组和空白对照组。cck-8法测各组细胞的增殖情况;Transwell小室模型测定各组BIU-87细胞的相对侵袭率与迁移率变化;Western-blot法测各组细胞Sphk1、ERK1/2与p-ERK1/2蛋白水平;ELISA方法检测各组细胞上清中MMP-2、MMP-9、VEGF及uPA的蛋白含量的表达;qRT-PCR测各组细胞Sph K1、MMP-2、MMP-9、uPA的mRNA水平;Matrigel三维培养法观察各组细胞血管生成拟态(vasculogenic mimicry,VM)形成能力。结果:SphK1激活剂可促进BIU-87细胞侵袭与迁移,同时明显增强细胞Sph K1、ERK1/2与p-ERK1/2的蛋白表达,并促进MMP-2、MMP-9及u PA的蛋白分泌。促进Matrigel构建的三维培养系统中VM的形成;明显增强VEGF蛋白表达。Sph K1抑制剂则能抑制BIU-87细胞侵袭与迁移,同时抑制Sph K1、ERK1/2与p-ERK1/2的蛋白表达,并抑制MMP-2、MMP-9及u PA的蛋白分泌;三维培养中不能形成管状VM;明显减弱VEGF蛋白表达。以患者临床资料为研究对象通过q RT-PCR和IHC方法检测患者膀胱癌组织和邻近的正常组织Sphk1的mRNA和蛋白质的表达,研究其差异,并分析其与膀胱癌临床病理特征的关系。结果:在37对膀胱癌组织及临近正常组织中测量发现癌组织中SphK1m RNA的表达显著高于癌旁正常组织中的表达;并发现153例膀胱癌病理组织切片中Sph K1蛋白表达与膀胱癌病理分期(P=0.045)、肿瘤病理分级(P0.001)显著相关。通过绘制Kaplan-Meier生存曲线发现高表达Sph K1患者的5年生存率显著下降(P0.001)。多变量Cox回归分析显示SphK1的高表达可以作为这种疾病的一种独立的不良预后因素。结论Sph K1在膀胱尿路上皮癌发生发展中发挥着重要的作用,其机制可能与激活信号ERK1/2通路从而促进MMP-2、MMP-9及u PA蛋白分泌有关,进而促进VEGF表达并增强肿瘤细胞的侵袭迁移能力而发挥作用。我们可以将Sph K1作为膀胱尿路上皮癌的诊断治疗之分子靶点。
[Abstract]:Bladder cancer (BC) is the most likely primary tumor in the urinary system, with high morbidity and mortality. The main treatment measures are intracavitary bladder tumor resection, radical cystectomy, conformal radiotherapy, local perfusion or systemic chemotherapy, but these measures are reviewed alone or after combined treatment. The clinical data analysis showed that from 2007 to 2012, there were 14260 cases of bladder cancer from 44 large department of Urology centers in our country, of which more than 70% of the patients received recurrent metastasis after receiving treatment, and 30% of the recurrent tumors were more malignant than before. Targeted therapy has gradually become a clinical hotspot in the comprehensive treatment of tumor. It is of certain practical significance whether the bladder cancer has a suitable molecular target and how to use the target to slow down the recurrence and progression of the tumor. Sphingosine (Sph) can regulate the intracellular signaling pathway to cause cell cycle arrest and promote cell apoptosis; Sphingosine 1-phosphate (S1P) has a functional.Cer/Sph that promotes cell proliferation and has a dynamic balance in the level of S1P in the cell, and their role is to promote death and a kind of growth promoting, just the opposite, water. Balance determines the balance of action. The balance of the action determines whether the cell's fate is alive or dead. This phenomenon is called "Sphingolipid-rheostat function". It is considered that the Sphingosine kinases (SphKs) is the most important group of enzymes that regulate this balance, and it can promote the sheath of death. Ammonia alcohol, ceramide is converted to sphingosine phosphate. By inhibiting the activity of this protein, an adjustable rheostat can play an antitumor role in the direction of cell death. It is found that Sph K1 can be used as a means of cancer treatment. Tumor cell invasion and metastasis are important factors affecting the prognosis of tumor patients. Cell metastasis needs to undergo cancer cell adhesion to basal cells, destruction of cell matrix adhesion, hydrolysis of extracellular matrix protein, and a series of processes of angiogenesis. Antitumor research is to study how to block this series of processes, and then block the transfer, invasion and diffusion of cancer cells, which are also the prevention and treatment of bladder cancer. In the process of cancer cell invasion and migration, the degradation of Matrix-metalloproteinase (MMPs) is not only necessary for tumor invasion and tissue remodeling in cancer cell migration, but also one of the changes in the early stage of tumor invasion. Malignant cells degrade the extracellular matrix through free MMPs and promote the neovascularization. The members of the matrix metalloproteinase family, MMP-2 and MMP-9, can degrade type IV collagen and then promote the degradation of the matrix membrane. In the invasion and metastasis of a variety of tumor cells, their expressions are elevated, and the two phenomena are related. The extracellular reaction kinase (extracellularresp Onsekinases, ERK) is a protein in the MAPK protein family, which has an enzyme activity. Its biological function is to control cell proliferation and differentiation, regulate cell apoptosis, determine cell malignant transformation, and maintain the expression of.Erk in cell morphology and the up-regulated activation state, which can detect.P-erk1/2 as erk1/2 in many human malignant tumors. The activation form is activated by phosphorylation of 202 - bit l- threonine (l-threonine, thr202) with 204 - bit tyrosine (tyrosine, tyr204) in erk1/2. The effect is stronger than erk1/2. The literature review found that sphk1/s1p activates the erk1/2 pathway in many tumors to promote cancer cell invasion and migration. The urokinase type fibrinolytic activator (uro) Kinase-typeplasminogenactivator, uPA) can activate plasma fibrinolytic enzyme into enzymes and can degrade extracellular matrix substances independently or through activated enzymes, activate MMPs, cause the release of tumor stimulating factors and VEGF to promote cell invasiveness and increase the migration ability of tumor cells. The erk1/2 pathway, through a series of signal transduction, amplification, and feedback, can reduce the expression of mmp-2/9 and uPA, and they cooperate to suppress tumor metastasis. This study is based on SphK1 as an object to determine whether the expression of SphK1 in BC patients is abnormal, its level and clinicopathological characteristics and the prognosis of the disease are No correlation, and the mechanism of cell level and molecular level. The specific way is to use the chemical drugs that regulate the expression of SphK1 protein in bladder cancer cell BIU-87, to observe the invasion and migration of BIU-87 in bladder tumor cells after treatment, and to determine the erk1/2 and perk1/2 protein, MMP-2, MMP-9, uPA and uPA in cell. The changes in the secretion of VEGF protein, from the cell and molecular level to determine whether sphingosine kinase 1 has the effect of changing the invasion and migration of human bladder cancer cells, its effect is enhanced or weakened, and corresponded to the clinicopathological results, which is a preliminary mechanism for the treatment of molecular swelling with sphingosine kinase 1 as a target. The purpose of this study is to explore the purpose of SPH. The role of K1 in the development of bladder urothelial carcinoma and its mechanism provide a theoretical basis for the diagnosis and treatment of SphK1 as a molecular target for the diagnosis and treatment of bladder urothelial carcinoma. Materials, methods and results are based on the phorbol12-myristate13-acetate (phorbol12-myristate13-acetate, PMA) of the 12- myrisate (phorbol12-myristate13-acetate, PMA) of the cells and molecules. Two methyl sphingosine (n-dimethyld-erythrosphingsine, DMS) was a SphK1 inhibitor; 0.9%nacl as a blank reagent, the BIU-87 cell line of bladder cancer was treated as the activation group. The proliferation of the cells in each group was measured by the.Cck-8 method in the inhibition group and the blank control group. The relative invasion rate and mobility of BIU-87 cells in each group were measured by the Transwell compartment model. The levels of Sphk1, ERK1/2 and p-ERK1/2 protein were measured by Western-blot, and the protein content of MMP-2, MMP-9, VEGF and uPA in the supernatant of each group was detected by ELISA. Results: SphK1 activator can promote the invasion and migration of BIU-87 cells, and obviously enhance the protein expression of Sph K1, ERK1/2 and p-ERK1/2, and promote the protein secretion of MMP-2, MMP-9 and u PA, and promote the formation of VM in the three-dimensional culture system constructed Matrigel. Cell invasion and migration, inhibit the protein expression of Sph K1, ERK1/2 and p-ERK1/2, and inhibit the protein secretion of MMP-2, MMP-9 and u PA, and can not form tubular VM in three-dimensional culture, and obviously weaken the expression of VEGF protein. The expression of mRNA and protein and its relationship with the clinicopathological features of bladder cancer were analyzed. Results: the expression of SphK1m RNA in the 37 bladder cancer tissues and adjacent normal tissues was found to be significantly higher than that in the normal tissues adjacent to the carcinoma, and the Sph K1 protein table in the pathological sections of the bladder cancer was found. The 5 year survival rate of the patients with high expression of Sph K1 was significantly decreased (P0.001) by plotting the Kaplan-Meier survival curve (P0.001). The multivariable Cox regression analysis showed that the high expression of SphK1 could be used as an independent adverse prognostic factor of this disease. Conclusion Sph K1 was found in the 5. Bladder urothelial carcinoma plays an important role in the development and development of the bladder urothelial carcinoma, which may be associated with the activation of the signal ERK1/2 pathway to promote the secretion of MMP-2, MMP-9 and u PA protein, thereby promoting the expression of VEGF and enhancing the invasion and migration of tumor cells. We can treat Sph K1 as a diagnosis and treatment of bladder urothelial carcinoma. Molecular targets.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.14

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