A-Raf-S214C突變體對MAPK信號通路的影響及2個A-Raf激酶結(jié)合蛋白的功能研究
發(fā)布時間：2018-07-28 13:58
【摘要】：癌癥每年要吞噬數(shù)百萬人的生命,給人們的生活帶來了極大的困擾。在所有的癌癥中,肺癌的危害最為嚴重,其發(fā)病率和致死率均列首位。肺癌嚴重威脅著我國人民的生命與健康,而且癌癥的治療花費巨大,治療周期較長,給家庭和社會帶來了沉重的負擔。因此,研究肺癌的發(fā)病機理,尋找治療肺癌的新靶點對人類的生命健康至關(guān)重要。Raf 激酶(Rapidly Accelerated Fibrosarcoma)是逆轉(zhuǎn)錄病毒致癌基因 v-Raf的同源蛋白,它是癌癥發(fā)生的關(guān)鍵激酶,它與肺癌的發(fā)病、治療和預(yù)后直接相關(guān)。A-Raf是Raf激酶家族成員之一,其生物學功能至今不清,可能參與了能量代謝的調(diào)節(jié)和細胞的轉(zhuǎn)化。此外,部分研究表明A-Raf的突變可導致肺癌、卵巢癌和大腸癌。有研究報道A-Raf-S214C突變體能轉(zhuǎn)化支氣管表皮細胞,使其發(fā)生癌變,在朗格漢氏增生癥中也發(fā)現(xiàn)該突變。因此,調(diào)控A-Raf激酶有望成為治療癌癥的重要手段。GRP78和PARP1均為A-Raf激酶的結(jié)合蛋白。前者參與內(nèi)質(zhì)網(wǎng)應(yīng)激,可維持內(nèi)質(zhì)網(wǎng)穩(wěn)態(tài),而且其在某些腫瘤細胞中呈高表達;后者主要參與DNA損傷修復(fù)和介導細胞凋亡。我們也可通過調(diào)控其表達,來研究其表達水平的上調(diào)、下調(diào)或者其活性被抑制時對腫瘤細胞的影響,尋找更為有效的治療腫瘤的新靶點。本研究主要對Raf激酶家族的三個成員(A-Raf,B-Raf和C-Raf)之一 A-Raf激酶進行系統(tǒng)研究。首先是在實驗室成功構(gòu)建A-Raf質(zhì)粒的基礎(chǔ)上,成功構(gòu)建了其突變體A-Raf S214C的質(zhì)粒及穩(wěn)定細胞系。接下來我們利用免疫共沉淀技術(shù)和質(zhì)譜技術(shù),得到A-Raf激酶結(jié)合蛋白譜。我們挑選出與腫瘤發(fā)生相關(guān)的結(jié)合蛋白GRP78和PARP1,通過siRNA、過表達、基因敲除等手段對其表達水平進行調(diào)控,研究其對肺癌細胞(A549)功能的影響。通過研究A-Raf S214C突變體對ERK信號作用,我們發(fā)現(xiàn),A-Raf S214C突變體是A-Raf的激活突變體,其對MAPK信號通路下游信號分子ERK的磷酸化能力較A-Raf更強。本研究還成功構(gòu)建了 A-Raf激酶結(jié)合蛋白GRP78和PARP1蛋白在A549細胞中的基因敲降穩(wěn)定細胞系和高表達穩(wěn)定細胞系。通過CCK8實驗、細胞劃痕實驗及Transwell實驗證明,敲降PARP1后的A549細抗凋亡能力增加,細胞的遷移能力也增加。這些結(jié)果為我們研究通過調(diào)控PARP1來治療癌癥提供了新的依據(jù)。本研究將為研究A-Raf激酶突變體的致癌機理提供新的線索,通過對Raf激酶結(jié)合蛋白深入研究,為治療A-Raf激酶相關(guān)的疾病,特別是以肺癌為代表的癌癥提供新的靶點和方法。
[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.
【學位授予單位】：昆明理工大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R734.2
本文編號：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.
【學位授予單位】：昆明理工大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R734.2
【參考文獻】
相關(guān)期刊論文 前1條
1 陸嘉德;郭曄;;腫瘤靶向治療新探:多靶點Raf激酶抑制劑[J];中國癌癥雜志;2007年01期
，本文編號：2150423
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