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研究生:林妍汎
研究生(外文):Yen-Fan Lin
論文名稱:探討ANKRD52對肺腺癌轉移的抑制作用
論文名稱(外文):Inhibition of lung adenocarcinoma metastasis by ANKRD52
指導教授:吳成文
指導教授(外文):Cheng-Wen, Wu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:英文
論文頁數:50
中文關鍵詞:肺腺癌
外文關鍵詞:lung adenocarcinomaankyrin repeat proteinsmetastasis
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根據國際世界衛生組織統計,肺癌一直是所有癌症中致死率最高的一種類型,肺癌中又以非小細胞肺癌佔多數,依據分類非小細胞肺癌又可以細分成肺腺癌、鱗狀細胞癌及大細胞癌。其中肺腺癌因發現不易,並且常常伴隨著癌細胞轉移的現象,在晚期肺腺癌病患發生癌細胞轉移的機率很高,而且會產生比較差的癒後情形。腫瘤轉移是限制病患存活時間的關鍵因素,癌細胞若沒有轉移發生則能有效治癒大部分癌症,所以研究癌細胞轉移機制有助於瞭解以及治療病患。本篇研究探討ANKRD52,它屬於蛋白磷酸酶6複合體的組成之一,在這個蛋白複合體中,它扮演著藉導蛋白質-蛋白質交互作用的角色。我們探討它在肺腺癌中所扮演的角色,經由線上臨床數據庫oncomine、肺腺癌病人檢體、實驗室肺腺癌細胞株以及動物實驗,都驗證了我們的假設:ANKRD52能降低肺腺癌細胞株的爬行能力,進而影響癌細胞轉移能力。接著我們深入去了解ANKRD52於肺腺癌細胞中的分子調控機制,發現它是藉由影響PAK1的轉譯後修飾(Post-translational modification)-磷酸化(Phosphorylation)程度,使減低PAK1的磷酸活化態,以影響肺腺癌細胞的爬行能力。最後,我們希望設計出一種以蛋白質結構為基礎的抑制方式,來減低藥物脫靶(off-target)情形。
According to WHO’s mortality data, lung cancer is one of the deadliest cancer type. Based on histological types, lung cancer is classified into two groups: small cell lung cancer and non-small cell lung cancer. Non-small cell lung cancer is the major type and adenocarcinoma occurs the most. As a leading cause of cancer-related deaths, metastasis is the ultimate challenge in our effort to fight cancer as a life-threatening disease.
A member of ankyrin repeat proteins, ankyrin repeat domain 52 (ANKRD52), is studied here to be involved in the regulation of lung adenocarcinoma metastasis. Ankyrin repeat proteins are known to function as a platform for protein-protein interactions and ANKRD52 was shown to interact with protein phosphatase 6 (PP6). This study aimed to investigate the anti-metastatic roles of ANKRD52 in lung adenocarcinoma. Immunohistochemistry staining in lung cancer tissue revealed reduced ANKRD52 expression in late-staged cases. Using lung cancer cell lines, ANKRD52 knockdowns showed significantly increased cell mobility while forced expressed ANKRD52 decreased cell mobility. Using mass spectrometry analysis, p21-activated kinases (PAK1) was identified to interact with ANKRD52. p21- activated kinases(PAK1) signaling is necessary to develop cancer progression that actives several oncogenic signaling pathways and PAK1 also promote tumor cell invasion and metastasis. Upregulated PAK1 phosphorylation was detected in ANKRD52 knockdowns and also in PP6 knockdowns. Our model demonstrated a novel anti-metastatic protein, ANKRD52, that regulates cell mobility through interactions with PP6 and dephosphorylation of PAK1. Besides, we attempt to design ankyrin repeat proteins to improve the off-target effects of PAK1 inhibitors.
致謝 i
Abstract ii
中文摘要 iv
Table of Content v
List of Figures vii
List of Tables viii
Chapter 1 . Introduction 1
1.1 Lung cancer 1
1.2 Tumor metastasis 1
1.3 Ankyrin Repeat Protein 3
1.4 Post-translational modification 3
1.4.1 Phosphorylation/ Dephosphorylation 4
1.5 Protein phosphatases 5
1.5.1 Protein phosphatase 6 5
1.6 p21-activated kinase (PAK) family - p21-activatied kinase 1 7
1.7 Working hypothesis 8
Chapter 2 . Materials and Methods 9
2.1 Plasmid construction 9
2.2 Cell culture 10
2.3 Transient transfection 10
2.4 Lentivirus generation, viral infection, stable cell line selection 11
2.5 RNA extraction and reverse transcription PCR 11
2.6 Quantitative real-time PCR 12
2.7 Protein extraction 13
2.8 Immunoblotting 13
2.9 Immunoprecipitation 14
2.10 Transwell migration assay and Invasion assay 15
2.11 Animal experiments - tail vein metastasis assay 15
2.12 Statistical analysis 16
Chapter 3 . Results 17
3.1 Reduced ANKRD52 mRNA expression level in stage IV lung adenocarcinoma patients. 17
3.2 ANKRD52 protein expression levels in CL-cell lines were negatively correlated with cell mobility. 18
3.3 Silencing of ANKRD52 increases cell mobility in vitro. 19
3.4 Raised ANKRD52 suppressed cell mobility in vitro. 19
3.5 Enhancement of ANKRD52 suppressed metastasis in animal model. 20
3.6 ANKRD52 associates with PAK1. 20
3.7 Reduction of ANKRD52 increases PAK1 phosphorylation level. 21
3.8 ANKRD52 is a unique regulatory subunit of PP6 to specifically recognizes PAK1. 22
3.9 ANKRD52 fragments interact with PAK1 in vitro. 23
Chapter 4 . Discussion 24
Chapter 5 . Conclusion 29
Figures 30
Tables 42
Reference 45
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