细胞骨架调节蛋白WASH对食管鳞癌进展的影响及其机制研究

发布时间：2018-03-16 10:26

本文选题：食管鳞癌　切入点：WASH　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景和目的食管癌是一种常见的消化道恶性肿瘤。近些年来,食管癌的发病率明显上升,因此针对食管癌发病机理的研究以及发现食管癌的治疗靶点迫在眉睫。在我国,食管癌患者的5年生存率不足20%,食管鳞癌的发病率占食管癌的90%,因此在本文主要是针对食管鳞癌的研究。越来越多的报道显示,细胞骨架的意义已经超出了只做为细胞形态结构的概念。近年研究发现,细胞的骨架结构及功能的改变也是导致肿瘤发生和发展的一个必不可少的重要因素。且在很多研究中表明,肿瘤细胞的恶性表型通常与肌动蛋白相关蛋白的异常表达相关。在本研究中,我们所关注的细胞骨架调节蛋白WASH,又叫WASHC1,是WASP(Wiskott-Aldrich syndrome Protein)家族中一种重要的细胞骨架调节相关蛋白。在肿瘤中,有关WASH细胞骨架调节蛋白参与肿瘤发生发展尚鲜见报道。作为一种重要的细胞骨架调节蛋白,WASH是否影响肿瘤细胞的命运,能否影响肿瘤细胞的进展和转移等等,这些问题都值得进一步的探讨和研究。本课题的主要任务是一方面鉴定WASH在食管鳞癌的发生发展中所起的重要作用;另一方面找到WASH影响食管鳞癌进展的机制,进一步结合临床探究其意义。方法收集84例食管鳞癌患者的癌组织及配对的癌旁组织,采用荧光定量PCR检测WASH在基因水平的表达,并分析WASH的表达与病人病理参数的相关性,以及其与病人生存相关性;利用免疫组化实验检测WASH在不同分期食管鳞癌肿瘤组织中的表达情况;在3株食管鳞癌细胞系中,检测WASH在食管鳞癌肿瘤干细胞和非干细胞的表达情况;利用基因沉默技术沉默WASH之后,探究食管鳞癌细胞的成球情况,通过荧光定量和westernblot检测部分干性基因的表达;通过瞬时转染技术过表达WASH之后,检测食管癌细胞系的成球情况及干性基因的表达情况;在利用sh RNA技术沉默WASH之后,多因子试剂盒、荧光定量PCR及ELISA检测细胞因子的表达差异,并探究其上下游机制;动物实验探究WASH在裸鼠体内的生长情况,探究WASH的表达对肿瘤进展的影响;为了探究WASH的上游机制,利用网站预测及双荧光报告等实验探究与其靶向的micro RNA,并在45例病人中检测此micro RNA的表达情况,检测与临床参数相关性,探究micro RNA与WASH的相关性,分析mircro RNA与病人预后相关性。结果1.荧光定量PCR结果显示:WASH在癌组织比癌旁组织高表达,差异有明显统计学意义。WASH的表达与病理分期有明显相关性。2.免疫组化结果显示:WASH在癌组织比癌旁组织高表达,差异有明显统计学意义。WASH的表达与病理分期及分化有明显相关性。且WASH与病人预后成负相关。3.成球实验结果显示:沉默WASH之后,食管鳞癌细胞系成球减少,差异具有统计学差异。且干性基因的表达也降低。瞬时过表达WASH之后,食管鳞癌细胞系成球增多,干性基因表达增加。4.多因子检测实验结果显示:沉默WASH后,细胞因子CXCL8的表达明显降低。差异具有统计学差异。5.动物实验结果显示:皮下注射稳定敲低WASH的细胞系,相对于对照组肿瘤体积及质量显著减少。在小鼠的肿瘤组织中,相对于对照组,敲低WASH后,CXCL8的表达显著降低。6.通过targetscan,mi Rbase等预测网站及利用双荧光报告实验检测出mi R-637靶向结合WASH的3’-UTR。并且在瞬时转染mi R-637之后,WASH的表达显著降低。并且通过构建mi R-637的稳定过表达系统,发现WASH的表达显著降低。在45例食管鳞癌临床病人中,mi R-637在癌旁组织比癌组织高表达,差异有明显统计学意义。在临床病人中,mi R-637表达与WASH的表达有显著负相关。结论1.WASH在食管鳞癌癌组织中是高表达的。WASH促进了食管鳞癌细胞的干性,促进了食管鳞癌的发生发展。2.在食管鳞癌中,低表达的mi R-637导致WASH的上调,进而WASH通过上调CXCL8促进食管鳞癌细胞的干性,促进食管鳞癌的进展。
[Abstract]:Background and objective: esophageal cancer is a common malignant tumor of digestive tract. In recent years, the incidence of esophageal cancer increased significantly, so the study on the pathogenesis of esophageal cancer and the discovery of therapeutic targets of esophageal cancer is imminent. In China, the 5 year survival rate of less than 20% of patients with esophageal cancer, the incidence rate of esophageal squamous cell carcinoma for esophageal cancer 90%, so in this paper is mainly study on esophageal squamous cell carcinoma. More and more reports indicate that the cytoskeleton has significance beyond just as the concept of cell morphology. Recent studies have found that skeleton structure and function of cells can also lead to the change of tumor occurrence and development of an indispensable factor. And that in many cases, the abnormal expression of malignant phenotype of tumor cells is associated with actin protein. In this study, we focus our attention on the regulation of cytoskeletal protein WASH , also known as WASHC1, WASP (Wiskott-Aldrich syndrome Protein) is a kind of important cytoskeletal associated protein family. In tumors, the WASH cytoskeletal protein involved in tumor development was rarely reported. As an important cytoskeletal protein, the influence of WASH on tumor cell fate, and can progress the impact of the transfer of tumor cells and so on, these issues are worthy of further study and research. The main task of this project is on the one hand in the identification of WASH plays an important role in the development of esophageal squamous cell carcinoma; on the other hand, to find the mechanism of WASH on the progression of esophageal squamous cell carcinoma, further explore the combined with clinical significance. Methods patients with esophageal squamous cell carcinoma 84 cases of cancer tissues and paired adjacent tissues, the expression by fluorescence quantitative PCR detection of WASH at the gene level, and analyze the expression and pathological parameters of the patients with WASH Correlation between patient survival and its correlation; expression by immunohistochemical assay of WASH in different stages of esophageal squamous cell carcinoma tissues; in 3 strains of esophageal squamous cell carcinoma cell lines, detection of WASH stem cells and the expression of non stem cells in esophageal squamous cell carcinoma tumor; after silencing WASH using gene silencing technology, a ball study of esophageal carcinoma cells by fluorescence quantitative detection expression of stemness genes and Westernblot; overexpression of WASH by transfection technique, ball and dry into gene detection of esophageal carcinoma cell line expression; after using WASH sh RNA silencing technology, multi factor kit, differential expression and fluorescence quantitative PCR ELISA detection of cytokines, and to explore its downstream mechanism; animal experiment research of WASH growth in nude mice, the expression of WASH on influence on tumor progression in order to exploration; The upstream mechanism of the WASH, using the site prediction and double fluorescence report and experiment inquiry targeted micro RNA, and the expression of micro RNA was detected in 45 patients, correlation detection and clinical parameters of micro and RNA, to explore the relevance of WASH, RNA and Mircro analysis of prognosis correlation. Results 1. fluorescent quantitative PCR the results showed that WASH high expression in cancer tissue than in adjacent tissues, the difference has statistical significance of.WASH expression and pathological stage there was significant correlation between.2. immunohistochemistry results showed that: the expression of WASH in cancer tissue than in adjacent tissue, there are significant differences in the expression of.WASH and pathological stage and differentiation and have significant correlation. The prognosis of WASH patients with a negative correlation of.3. into the ball experiment results showed that silencing of WASH, esophageal squamous cell carcinoma cell lines into the ball is reduced, a statistically significant difference. The expression of genes is reduced. And the dry instant After overexpression of WASH in esophageal squamous cell carcinoma cell line, into the ball more, dry gene expression increased.4. results in multiple factor detection experiments showed that silencing of WASH and cytokine CXCL8 expression significantly decreased. A statistically significant difference.5. animal experiment results showed that subcutaneous injection of stable knockdown of WASH cell line, tumor volume and relative to the control group the quality was significantly reduced in mice. The tumor tissues, compared with the control group, knockdown of WASH, the expression of CXCL8 was significantly reduced by.6. targetscan, MI Rbase and predict the site with dual luciferase reporter experiments detected mi R-637 target WASH 3 '-UTR. and R-637 MI after transfection, the expression of WASH significantly reduced. And through the construction of MI R-637 stable expression system, expression of WASH decreased significantly. In 45 patients of esophageal squamous cell carcinoma, MI R-637 in cancer tissues than in cancer tissues with high expression And there was a significant difference. In clinical patients, MI expression of R-637 and WASH have significant negative correlation. Conclusion 1.WASH in esophageal squamous cell carcinoma is high expression of.WASH promoted esophageal squamous cell dry, promote the occurrence and development of esophageal squamous cell carcinoma.2. in esophageal squamous carcinoma, low expression of MI R-637 leads to the upregulation of WASH and WASH, through the upregulation of CXCL8 promote esophageal squamous cell carcinoma and promoting the progress of the dry, esophageal squamous cell carcinoma.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1

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