TLE4促进结直肠癌细胞增殖和迁移的作用及分子机制研究

发布时间：2018-05-16 16:05

本文选题：TLE4 + 结直肠癌　；参考：《南方医科大学》2017年博士论文

【摘要】：研究背景和目的:结直肠癌(colorectal cancer,CRC)是最常见的消化系统恶性肿瘤之一。据报道,全球结直肠癌每年新发病例数约120万,发病率居所有恶性肿瘤中的第3位,病死率居第4位。此外,随着人们生活方式和饮食结构的转变,其发病率呈逐年上升趋势,且发病年龄呈年轻化特点。转移是引起CRC患者死亡的首要原因,大多数病人在确诊时就已发生淋巴结转移或远处转移。因此,深入探讨结直肠癌发生发展及转移的分子机制,寻找早期诊断、早期治疗和预防的有效途径,始终是结直肠癌研究领域的重要内容。TLE4基因是Groucho/Tle基因家族的重要成员之一,Groucho/Tle蛋白在胚胎发育和形态发生过程中发挥重要作用,可调控相关关键信号通路的活性,包括受体酪氨酸激酶/Ras/MAPK、Notch、Wnt、Hedgehog、JNK等信号通路。据报道,TLE4在造血功能、骨发育过程中起着重要作用,有研究提示TLE4在急性髓性白血病(acute myelogenous leukemia,AML)中起着抑制作用。在白血病细胞株中下调TLE4表达可以增加白血病细胞的分裂,过表达TLE4可引起细胞凋亡的增加。然而,关于TLE4在包括结直肠癌在内的实体肿瘤中的作用知之甚少。我们前期的生物信息学分析显示,TLE4在结直肠癌组织中表达水平显著高于配对的癌旁正常肠粘膜组织;但是,TLE4在结直肠癌发生和演进过程中的作用及分子机制目前尚不清楚。本课题拟检测TLE4蛋白在结直肠癌中的表达情况,分析其表达与结直肠癌患者临床病理参数及预后之间的关系,并通过多种体内外实验研究TLE4在结直肠癌发生、发展和转移过程中的作用及相关分子机制,以期为结直肠癌的个体化诊疗和靶向药物的开发提供一定的理论依据。方法:1.运用公共数据库(GEO)数据分析TLE4在结直肠癌中的表达情况;2.利用Western blot和实时定量PCR技术检测TLE4在新鲜结直肠癌和配对癌旁正常组织标本中的表达情况;通过免疫组化技术检测结直肠癌患者石蜡组织标本中TLE4的表达,分析其与结直肠癌患者临床病理特征及预后间的相关性;3.通过分子克隆技术构建TLE4稳定过表达和干扰表达的结直肠癌细胞株,并通过实时定量PCR和Western blot技术检测TLE4和相关信号通路蛋白在稳定细胞株中mRNA和蛋白水平的表达情况;4.通过平板克隆形成实验、软琼脂克隆形成实验、MTT实验体外检测TLE4稳定过表达和干扰对结直肠癌细胞增殖能力的影响,通过Transwell实验体外检测TLE4稳定过表达和干扰对结直肠癌细胞迁移能力的影响;利用裸鼠皮下成瘤模型,并运用免疫组化实验体内检测TLE4稳定过表达和干扰对结直肠癌细胞增殖能力的影响;5.实时定量PCR和Western blot技术检测TLE4稳定过表达和干扰后结直肠癌细胞中JNK/c-Jun通路及其相应靶点分子的变化;6.使用 JNK 抑制剂 SP600125 处理 SW480/TLE4 和 HT29/TLE4 细胞 24 小时后,进一步通过Western blot检测JNK/c-Jun通路及其相应靶点分子的变化,通过MTT实验、平板克隆形成实验、软琼脂克隆形成实验和Transwell实验检测结直肠癌细胞的增殖及迁移能力。结果:(数据使用SPSS 20.0 for Windows统计软件进行分析)1.TLE4在结直肠癌组织中表达水平较配对癌旁正常组织显著升高;其表达水平与患者Dukes分期、淋巴结转移等临床病理参数之间呈正相关关系:2.Kaplan-Meier法分析结果显示,TLE4高表达组的总体生存率显著低于低表达组,与患者的生存预后呈负相关关系;3.TLE4的过表达促进结直肠癌细胞的增殖、迁移和裸鼠皮下成瘤能力,而干扰TLE4表达后则抑制结直肠癌细胞的增殖、迁移和裸鼠皮下成瘤能力;4.TLE4能激活JNK/c-Jun分子信号通路的活性,使用JNK/c-Jun分子信号通路阻滞剂SP600125能够抵消TLE4促进结直肠癌细胞增殖和迁移的作用。TLE4可能通过激活JNK/c-Jun分子信号通路促进结直肠癌细胞增殖和迁移。结论:TLE4在结直肠癌组织中显著上调,其表达上调能够通过激活JNK/c-Jun信号通路促进结直肠癌细胞的增殖及迁移。
[Abstract]:Background and purpose: colorectal cancer (CRC) is one of the most common malignant tumors of the digestive system. It is reported that the number of new cases of colorectal cancer is about 1 million 200 thousand in the world, the incidence is the third in all malignant tumors, and the fatality rate is fourth. In addition, the incidence of the disease is with the change of lifestyle and diet structure. There is a trend of increasing trend year by year, and the age of onset is young. Metastasis is the primary cause of death in CRC patients. Most patients have lymph node metastasis or distant metastasis at the time of diagnosis. Therefore, the molecular mechanism of the development and metastasis of colorectal cancer is explored, and the effective ways to find early diagnosis, early treatment and prevention are always sought. .TLE4 gene is one of the important members of the field of colorectal cancer research. The gene is one of the important members of the Groucho/Tle gene family. Groucho/Tle protein plays an important role in the process of embryonic development and morphogenesis. It can regulate the activity of related key signaling pathways, including receptor tyrosine kinase / Ras/MAPK, Notch, Wnt, Hedgehog, JNK and other signaling pathways. It is reported that TLE4 plays an important role in hematopoiesis and bone development. Studies have suggested that TLE4 plays an inhibitory role in acute myelogenous leukemia (acute myelogenous leukemia, AML). The downregulation of TLE4 in leukemia cell lines can increase the division of leukemia cells, and the over expression of TLE4 may cause an increase in apoptosis. However, TLE4 The role of TLE4 in colorectal cancer tissue is significantly higher than that of normal paracancerous intestinal mucosa in colorectal cancer. However, the role and molecular mechanism of TLE4 in the development and evolution of colorectal cancer is not clear. To examine the expression of TLE4 protein in colorectal cancer, analyze the relationship between its expression and the clinicopathological parameters and prognosis of colorectal cancer patients, and to study the role of TLE4 in the process of colorectal cancer, development and metastasis through a variety of in vivo and in vivo experiments, as well as the related molecular mechanism, in order to be an individualized diagnosis and target for colorectal cancer. To provide some theoretical basis for the development of drugs. Methods: 1. the expression of TLE4 in colorectal cancer was analyzed by using public database (GEO) data. 2. the expression of TLE4 in normal tissue specimens of fresh colorectal cancer and paired cancerous tissues was detected by Western blot and real-time quantitative PCR technique; and the colorectal cancer was detected by immunohistochemical technique. The expression of TLE4 in paraffin tissue specimens of cancer patients was analyzed, and the correlation between the clinicopathological features and prognosis of colorectal cancer patients was analyzed. 3. the colorectal cancer cell lines with TLE4 stable overexpression and interference expression were constructed by molecular cloning technique, and the stability of TLE4 and related signal pathway proteins was determined by real-time quantitative PCR and Western blot technique. The expression of mRNA and protein levels in cell lines; 4. through the formation of flat clones, the formation of soft agar cloning, and the effect of TLE4 stable overexpression and interference on the proliferation of colorectal cancer cells in vitro, and the stability of TLE4 over expression and interference to the migration of colorectal cancer cells in vitro by the Transwell test. Effect; using the subcutaneous tumor model of nude mice, and the effect of TLE4 stable overexpression and interference on the proliferation of colorectal cancer cells in the immuno histochemical experiment; 5. real-time quantitative PCR and Western blot techniques for detecting the changes of JNK/ c-Jun pathway and the corresponding target molecules in colorectal cancer cells after the stable overexpression and interference of TLE4; 6. After treating SW480/TLE4 and HT29/TLE4 cells with JNK inhibitor SP600125 for 24 hours, the changes in the JNK/c-Jun pathway and the corresponding target molecules were detected by Western blot. The proliferation and migration ability of the colorectal cancer cells were detected by MTT experiment, the formation of flat clones, the cloning of soft agar and the ability to detect the migration of the colorectal cancer cells. Fruit: (data was analyzed using SPSS 20 for Windows statistical software) the expression level of 1.TLE4 in colorectal cancer tissues was significantly higher than that of normal para cancerous tissue, and the expression level was positively correlated with the clinicopathological parameters such as Dukes staging and lymph node metastasis. The results of 2.Kaplan-Meier analysis showed that the high expression group of TLE4 was found. The overall survival rate was significantly lower than that of the low expression group, which had a negative correlation with the survival prognosis of the patients. The overexpression of 3.TLE4 promoted the proliferation, migration and subcutaneous tumor formation of the colorectal cancer cells, while the interference of TLE4 expression inhibited the proliferation of colorectal cancer cells, migration and subcutaneous tumor formation ability of nude mice; 4.TLE4 could activate the JNK/c-Jun molecular signal. The activity of the pathway, the use of the JNK/c-Jun molecular signaling pathway blocker SP600125 can counteract the effect of TLE4 on the proliferation and migration of colorectal cancer cells..TLE4 may promote the proliferation and migration of colorectal cancer cells by activating the JNK/c-Jun molecular signaling pathway. Conclusion: TLE4 is significantly up-regulated in the colorectal cancer group, and its up regulation can be induced by excitation. Living JNK/c-Jun signaling pathway promotes proliferation and migration of colorectal cancer cells.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.3

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