BRCA1结合蛋白BRAP促进食管癌侵袭转移

发布时间：2018-05-06 09:17

本文选题：BRAP + 转移　；参考：《北京协和医学院》2017年博士论文

【摘要】：背景和目的:肿瘤转移是癌症患者死亡的主要原因,但其分子机制并未完全阐明。多项研究已经表明与肿瘤发生相关的基因也可能影响肿瘤的转移。因此本课题研究的目的是为了阐明实验室前期通过全基因组关联研究(Genome-wide association study,GWAS)发现的易感基因能否影响肿瘤的转移。方法:首先我们运用高通量siRNA文库结合细胞迁移实验对食管癌11个易感区段内的47个基因进行筛选及功能注释。利用慢病毒载体在食管癌细胞(KYSE30和KYSE150)以及HeLa细胞中将迁移相关基因进行敲降和过表达,体外分析扰动其表达对细胞迁移和侵袭能力的影响。另外我们通过裸鼠尾静脉注射癌细胞构建裸鼠肺转移瘤模型,体内分析敲降其表达对肿瘤细胞在裸鼠肺部定植的影响。进一步我们通过荧光素酶报告基因系统、免疫荧光、免疫印迹以及免疫共沉淀等实验,分析易感基因影响肿瘤细胞转移的分子机制。另外我们采用实时定量PCR、免疫印迹以及免疫组化的方法,从mRNA水平和蛋白水平上检测易感基因在食管癌组织以及癌旁组织中的表达情况,并通过Log-rank生存分析的方法,分析其表达和患者生存的关系。结果:通过高通量筛选,我们发现BRAP(BRCA1结合蛋白)是一个肿瘤细胞迁移相关基因,敲降BRAP的表达能够降低肿瘤细胞的迁移侵袭能力以及肿瘤细胞在裸鼠肺部的定植。高表达BRAP能够促进肿瘤细胞的迁移侵袭能力,其机制是通过促进蛋白激酶PKCα和IKKβ的结合,使IKKβ发生磷酸化,进而激活NF-κB信号通路,最终促进NF-κB靶基因MMP9、VEGFC的表达。另外我们发现DNA水平拷贝数的增加影响BRAP在食管癌中高表达,且高表达患者的生存时间显著低于低表达患者的生存时间。结论:在食管癌细胞和组织中,BRAP的高表达能够促进食管癌细胞的侵袭转移能力,降低患者的生存时间。高表达BRAP的细胞能够通过激活NF-κB信号通路,上调MMP9和VEGFC的表达,促进裸鼠肺部转移瘤的形成。因此,BRAP可能成为食管癌治疗的靶点。
[Abstract]:Background and objective: tumor metastasis is the main cause of death in cancer patients, but its molecular mechanism is not fully elucidated. Several studies have shown that genes associated with tumorigenesis may also affect tumor metastasis. Therefore, the purpose of this study is to elucidate whether the susceptibility genes discovered by Genome-wide association studyGWASin the early stage of the laboratory can affect the metastasis of tumor. Methods: first, 47 genes in 11 susceptible regions of esophageal cancer were screened and annotated by high throughput siRNA library and cell migration assay. Lentivirus vectors were used to knockdown and overexpression the genes associated with migration in esophageal carcinoma cell line KYSE30 and KYSE150) and to analyze the effect of perturbed expression on cell migration and invasion in vitro. In addition, the lung metastasis model of nude mice was established by injecting cancer cells into tail vein of nude mice, and the effect of knockout on tumor cell colonization in nude mice lung was analyzed in vivo. Furthermore, we analyzed the molecular mechanism of susceptibility genes affecting tumor cell metastasis by luciferase reporter gene system, immunofluorescence, immunoblotting and immunoprecipitation. In addition, we used real-time quantitative PCR, Western blot and immunohistochemical methods to detect the expression of susceptible genes in esophageal carcinoma and adjacent tissues from the level of mRNA and protein, and by means of Log-rank survival analysis. The relationship between its expression and patient survival was analyzed. Results: through high throughput screening, we found that BRAP(BRCA1 binding protein is a gene associated with tumor cell migration. The expression of knockdown B RAP can reduce the migration and invasion of tumor cells and the colonization of tumor cells in the lung of nude mice. Overexpression of BRAP can promote the migration and invasion ability of tumor cells. The mechanism is that IKK 尾 is phosphorylated by promoting the binding of protein kinase PKC 伪 and IKK 尾, thus activating NF- 魏 B signaling pathway, and finally promoting the expression of NF- 魏 B target gene MMP9 and VEGFC. In addition, we found that the increase of DNA horizontal copy number affected the high expression of BRAP in esophageal carcinoma, and the survival time of patients with high expression was significantly lower than that of patients with low expression. Conclusion: the high expression of BRAP in esophageal carcinoma cells and tissues can promote the invasion and metastasis ability of esophageal cancer cells and reduce the survival time of the patients. The cells with high expression of BRAP can up-regulate the expression of MMP9 and VEGFC by activating NF- 魏 B signaling pathway and promote the formation of lung metastases in nude mice. Therefore, BRAP may be a target for the treatment of esophageal cancer.
【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】：2017
【分类号】：R735.1

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