A-Raf-S214C突变体对MAPK信号通路的影响及2个A-Raf激酶结合蛋白的功能研究
发布时间：2018-07-28 13:58
【摘要】：癌症每年要吞噬数百万人的生命,给人们的生活带来了极大的困扰。在所有的癌症中,肺癌的危害最为严重,其发病率和致死率均列首位。肺癌严重威胁着我国人民的生命与健康,而且癌症的治疗花费巨大,治疗周期较长,给家庭和社会带来了沉重的负担。因此,研究肺癌的发病机理,寻找治疗肺癌的新靶点对人类的生命健康至关重要。Raf 激酶(Rapidly Accelerated Fibrosarcoma)是逆转录病毒致癌基因 v-Raf的同源蛋白,它是癌症发生的关键激酶,它与肺癌的发病、治疗和预后直接相关。A-Raf是Raf激酶家族成员之一,其生物学功能至今不清,可能参与了能量代谢的调节和细胞的转化。此外,部分研究表明A-Raf的突变可导致肺癌、卵巢癌和大肠癌。有研究报道A-Raf-S214C突变体能转化支气管表皮细胞,使其发生癌变,在朗格汉氏增生症中也发现该突变。因此,调控A-Raf激酶有望成为治疗癌症的重要手段。GRP78和PARP1均为A-Raf激酶的结合蛋白。前者参与内质网应激,可维持内质网稳态,而且其在某些肿瘤细胞中呈高表达;后者主要参与DNA损伤修复和介导细胞凋亡。我们也可通过调控其表达,来研究其表达水平的上调、下调或者其活性被抑制时对肿瘤细胞的影响,寻找更为有效的治疗肿瘤的新靶点。本研究主要对Raf激酶家族的三个成员(A-Raf,B-Raf和C-Raf)之一 A-Raf激酶进行系统研究。首先是在实验室成功构建A-Raf质粒的基础上,成功构建了其突变体A-Raf S214C的质粒及稳定细胞系。接下来我们利用免疫共沉淀技术和质谱技术,得到A-Raf激酶结合蛋白谱。我们挑选出与肿瘤发生相关的结合蛋白GRP78和PARP1,通过siRNA、过表达、基因敲除等手段对其表达水平进行调控,研究其对肺癌细胞(A549)功能的影响。通过研究A-Raf S214C突变体对ERK信号作用,我们发现,A-Raf S214C突变体是A-Raf的激活突变体,其对MAPK信号通路下游信号分子ERK的磷酸化能力较A-Raf更强。本研究还成功构建了 A-Raf激酶结合蛋白GRP78和PARP1蛋白在A549细胞中的基因敲降稳定细胞系和高表达稳定细胞系。通过CCK8实验、细胞划痕实验及Transwell实验证明,敲降PARP1后的A549细抗凋亡能力增加,细胞的迁移能力也增加。这些结果为我们研究通过调控PARP1来治疗癌症提供了新的依据。本研究将为研究A-Raf激酶突变体的致癌机理提供新的线索,通过对Raf激酶结合蛋白深入研究,为治疗A-Raf激酶相关的疾病,特别是以肺癌为代表的癌症提供新的靶点和方法。
[Abstract]:Cancer engulfs millions of people every year, causing great trouble to people's lives. Of all cancers, lung cancer is the most serious, with the highest morbidity and mortality. Lung cancer is a serious threat to the lives and health of the people in our country, and the treatment of cancer costs a lot and the treatment period is long, which brings a heavy burden to the family and society. Therefore, to study the pathogenesis of lung cancer and to find new targets for the treatment of lung cancer are very important for human life and health. Raf kinase (Rapidly Accelerated Fibrosarcoma) is the homologous protein of retrovirus oncogene v-Raf, which is the key kinase in carcinogenesis. A-Raf is a member of Raf kinase family, and its biological function is still unclear, which may be involved in the regulation of energy metabolism and cell transformation. In addition, some studies have shown that mutations in A-Raf can lead to lung, ovarian and colorectal cancer. It has been reported that A-Raf-S214C mutagenesis can transform bronchial epidermis cells and cause carcinogenesis, which is also found in Langerhan's hyperplasia. Therefore, the regulation of A-Raf kinase is expected to be an important therapy for cancer. GRP78 and PARP1 are both binding proteins of A-Raf kinase. The former is involved in endoplasmic reticulum stress, maintaining endoplasmic reticulum homeostasis, and it is highly expressed in some tumor cells, while the latter is mainly involved in the repair of DNA damage and mediated apoptosis. We can also study the effect of up-regulation, down-regulation or inhibition of its activity on tumor cells by regulating its expression to find a more effective target for the treatment of tumor. In this study, A-Raf kinases, one of the three members of the Raf kinase family (A-Rafan B-Raf and C-Raf), were systematically studied. Firstly, on the basis of successfully constructing A-Raf plasmid in laboratory, the plasmid and stable cell line of its mutant A-Raf S214C were successfully constructed. Then we obtained A-Raf kinase binding protein profile by immunoprecipitation and mass spectrometry. We selected the binding proteins GRP78 and PARP1 associated with tumorigenesis and regulated their expression levels by siRNA-overexpression and gene knockout to study their effects on the function of lung cancer cells (A549). By studying the effect of A-Raf S214C mutants on ERK signals, we found that the A-Raf S214C mutants are active mutants of A-Raf, and their phosphorylation ability to the downstream signal molecule ERK of MAPK signaling pathway is stronger than that of A-Raf. The stable cell lines of gene knockdown and high expression of A-Raf kinase binding protein GRP78 and PARP1 protein were successfully constructed in A549 cells. The results of CCK8 assay, cell scratch test and Transwell test showed that the ability of fine anti-apoptosis and migration of A549 after knock down PARP1 increased. These results provide a new basis for our research on the treatment of cancer by regulating PARP1. This study will provide new clues for studying the carcinogenic mechanism of A-Raf kinase mutants, and provide new targets and methods for the treatment of A-Raf kinase-related diseases, especially lung cancer, through the in-depth study of Raf kinase binding proteins.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
本文编号：2150423
[Abstract]:Cancer engulfs millions of people every year, causing great trouble to people's lives. Of all cancers, lung cancer is the most serious, with the highest morbidity and mortality. Lung cancer is a serious threat to the lives and health of the people in our country, and the treatment of cancer costs a lot and the treatment period is long, which brings a heavy burden to the family and society. Therefore, to study the pathogenesis of lung cancer and to find new targets for the treatment of lung cancer are very important for human life and health. Raf kinase (Rapidly Accelerated Fibrosarcoma) is the homologous protein of retrovirus oncogene v-Raf, which is the key kinase in carcinogenesis. A-Raf is a member of Raf kinase family, and its biological function is still unclear, which may be involved in the regulation of energy metabolism and cell transformation. In addition, some studies have shown that mutations in A-Raf can lead to lung, ovarian and colorectal cancer. It has been reported that A-Raf-S214C mutagenesis can transform bronchial epidermis cells and cause carcinogenesis, which is also found in Langerhan's hyperplasia. Therefore, the regulation of A-Raf kinase is expected to be an important therapy for cancer. GRP78 and PARP1 are both binding proteins of A-Raf kinase. The former is involved in endoplasmic reticulum stress, maintaining endoplasmic reticulum homeostasis, and it is highly expressed in some tumor cells, while the latter is mainly involved in the repair of DNA damage and mediated apoptosis. We can also study the effect of up-regulation, down-regulation or inhibition of its activity on tumor cells by regulating its expression to find a more effective target for the treatment of tumor. In this study, A-Raf kinases, one of the three members of the Raf kinase family (A-Rafan B-Raf and C-Raf), were systematically studied. Firstly, on the basis of successfully constructing A-Raf plasmid in laboratory, the plasmid and stable cell line of its mutant A-Raf S214C were successfully constructed. Then we obtained A-Raf kinase binding protein profile by immunoprecipitation and mass spectrometry. We selected the binding proteins GRP78 and PARP1 associated with tumorigenesis and regulated their expression levels by siRNA-overexpression and gene knockout to study their effects on the function of lung cancer cells (A549). By studying the effect of A-Raf S214C mutants on ERK signals, we found that the A-Raf S214C mutants are active mutants of A-Raf, and their phosphorylation ability to the downstream signal molecule ERK of MAPK signaling pathway is stronger than that of A-Raf. The stable cell lines of gene knockdown and high expression of A-Raf kinase binding protein GRP78 and PARP1 protein were successfully constructed in A549 cells. The results of CCK8 assay, cell scratch test and Transwell test showed that the ability of fine anti-apoptosis and migration of A549 after knock down PARP1 increased. These results provide a new basis for our research on the treatment of cancer by regulating PARP1. This study will provide new clues for studying the carcinogenic mechanism of A-Raf kinase mutants, and provide new targets and methods for the treatment of A-Raf kinase-related diseases, especially lung cancer, through the in-depth study of Raf kinase binding proteins.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
【参考文献】
相关期刊论文 前1条
1 陆嘉德;郭晔;;肿瘤靶向治疗新探:多靶点Raf激酶抑制剂[J];中国癌症杂志;2007年01期
，本文编号：2150423
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