跳到主要內容

臺灣博碩士論文加值系統

(44.200.86.95) 您好!臺灣時間:2024/05/25 16:42
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:陳瑋庭
研究生(外文):Wei-Ting Chen
論文名稱:以定量磷酸化蛋白質體學探討鋅指蛋白ZNF322A在人類肺癌A549細胞中的調控路徑
論文名稱(外文):Quantitative Phosphoproteomic Analysis Reveals the Regulatory Pathways of ZNF322A in Human Lung Adenocarcinoma A549 Cells
指導教授:阮雪芬阮雪芬引用關係
指導教授(外文):Hsueh-Fen Juan
口試委員:黃宣誠王憶卿陳頌方李岳倫
口試委員(外文):Hsuan-Cheng HuangYi-Ching WangSung-Fang ChenYueh-Luen Lee
口試日期:2015-07-28
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:95
中文關鍵詞:鋅指蛋白致癌基因磷酸化蛋白質體學羥基酸修正金屬氧化物層析自噬作用
外文關鍵詞:Zinc-finger proteinoncogenephosphoproteomeHAMMOCautophagy
相關次數:
  • 被引用被引用:0
  • 點閱點閱:167
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
鋅指蛋白322A (ZNF322A) 是屬於C2H2型鋅指蛋白家族並為一種轉錄因子。近年的研究指出ZNF322A在肺癌病患與腫瘤發生有關,是一個致癌基因。然而,ZNF322A在肺癌細胞中所調控的下游訊息傳遞路徑尚未被了解透徹。我們利用羥基酸修正金屬氧化物層析(hydroxy acid-modified metal oxide chromatography, HAMMOC)及奈米級液相層析與串聯式質譜儀(nanoLC-MS/MS)闡述了在肺癌細胞中由ZNF322A下游訊息引發的蛋白質磷酸化現象。在本研究中,共發現了4501個磷酸化位置對應於1309個磷酸化蛋白質,其中有443個磷酸化位置顯著被ZNF322A所調控。利用生物資訊學研究法,包含功能富集分析,生物網路分析及磷酸化基序分析,我們提出先前尚未發現ZNF322A與哺乳動物雷帕黴素靶蛋白(mTOR)的訊息路徑及細胞自噬作用的相關性,並且展現了ZNF322A在癌症形成與細胞骨架調節等腫瘤相關形成過程中扮演重要的角色。本研究不只給予ZNF322A在後轉譯階層的分子調控資訊,也提供肺癌治療上一個新的方向。

ZNF322A is a transcription factor and belongs to the krüppel C2H2-type zinc-finger protein family. Recently, ZNF322A was reported as a potential oncogene in lung cancer patients and is critical for tumorigenesis. However, ZNF322A-mediated downstream signaling pathways in lung cancer cells remain unclear. Using hydroxy acid-modified metal oxide chromatography (HAMMOC) and nanoscale liquid chromatography–tandem MS (nanoLC-MS/MS), we examined protein phosphorylation induced by ZNF322A signaling in lung cancer A549 cells. We identified 4501 phosphorylation sites in 1309 phosphoproteins. Among these phosphosites, 443 were significantly changed in response to ZNF322A silencing. Using bioinformatics approaches including functional enrichments, network analysis and phosphorylation motif analysis, we highlighted a previously unidentified ZNF322A-mTOR signaling pathway and autophagic process. On the other hand, we demonstrated that ZNF322A plays an important role in cancer progression such as cytoskeleton regulation and tumor formation-associated process. This study not only gives new information about the molecular regulation by ZNF322A at post-translational level, but also provides a resource for the study of lung cancer therapy.

口試委員會審定書 i
誌謝 ii
中文摘要 iii
ABSTRACT iv
ABBRRVIATION v
CONTENTS vii
LIST OF FIGURES x
LIST OF TABLES xi
Chapter 1 INTRODUCTION 1
1.1 Zinc finger proteins 1
1.1.1 Global introduction 1
1.1.2 ZNF322A 2
1.2 Non-small cell lung cancer 3
1.3 Phosphoteomic study 3
1.3.1 Introduction 3
1.3.2 mTOR pathway 4
1.3.3 Autophagy 5
Chapter 2 EXPERIMENTAL PROCESSES 8
2.1 Cell culture 8
2.2 Cell transfection 8
2.3 RNA extraction and cDNA synthesis 8
2.3 Real-time quantitative RT-PCR (qRT-PCR) assays 9
2.4 Stage tip preparation 9
2.5 Sample preparation and extraction 10
2.6 Phosphoproteome sample preparation 11
2.6.1 Dimethyl labeling of peptides 12
2.6.2 Desalting with SDB-XC StageTips 12
2.6.3 Phosphopeptide enrichment with HAMMOC and fractionation 13
2.7 NanoLC–MS/MS analysis 14
2.8 Data analysis for phosphoproteomes 15
2.9 Functional annotation and clustering analyses 16
2.10 Western blot 17
2.11 TEM analysis of silencing A549 Cells 18
Chapter 3 RESULTS 19
3.1 The expression changes of ZNF322A in siRNA-transfected cells 19
3.2 Quantitative analysis of dynamic phosphorylation profiles regulated by ZNF322A in lung cancer A549 cells. 19
3.3 Biological functions and enrichment pathways regulated by ZNF322A in lung cancer cells. 21
3.4 Silencing ZNF322A induced A549 cell autophagy. 23
Chapter 4 DISCUSSION 25
Chapter 5 CONCLUSION 29
References 30
Figures 39
Tables 55
Appendix 94


(1) Lander, E.; Linton, L.; B., B., Initial sequencing and analysis of the human genome. Nature 2001, 409, 860-921.
(2) Schuh, R.; Aicher, W.; Gaul, U.; Côté, S.; Preiss, A.; Maier, D.; Seifert, E.; Nauber, U.; Schröder, C.; Kemler, R., A conserved family of nuclear proteins containing structural elements of the finger protein encoded by Krüppel, a Drosophila segmentation gene. Cell 1986, 47 (6), 1025-1032.
(3) Messina, D.; Glasscock, J.; Gish, W.; Lovett, M., An ORFeome-based analysis of human transcription factor genes and the construction of a microarray to interrogate their expression. Genome Res. 2004, 14 (10B), 2041-2047.
(4) Miller, J.; McLachlan, A.; Klug, A., Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985, 4 (6), 1609-1614.
(5) Tian, C.; Xing, G.; Xie, P.; Lu, K.; Nie, J.; Wang, J.; Li, L.; Gao, M.; Zhang, L.; He, F., KRAB-type zinc-finger protein Apak specifically regulates p53-dependent apoptosis. Nat. Cell Biol. 2009, 11 (5), 580-591.
(6) Jheon, A.; Ganss, B.; Cheifetz, S.; Sodek, J., Characterization of a Novel KRAB/C2H2 Zinc Finger Transcription Factor Involved in Bone Development. J. Biol. Chem. 2001, 276 (21), 18282-18289.
(7) Susanne Wagner, M. A.; Hess, P. O.; Hanso, J.; Malandro, H.; Hu, M.; Chen, R.; Kehrer, M.; Frodsham, C.; Schumacher, M.; Beluch, C.; Honer, M.; Skolnick, D. B.; B., B. R., A Broad Role for the Zinc Finger Protein ZNF202 in Human Lipid Metabolism. J. Biol. Chem. 2002, 275 (21), 15685-15690.
(8) Lai, K. P.; Chen, J.; He, M.; Ching, A. K.; Lau, C.; Lai, P. B.; To, K. F.; Wong, N., Overexpression of ZFX confers self-renewal and chemoresistance properties in hepatocellular carcinoma. Int. J. Cancer 2014, 135 (8), 1790-1799.
(9) Zhang, L.; Xu, Z.; Xu, X.; Zhang, B.; Wu, H.; Wang, M.; Zhang, X.; Yang, T.; Cai, J.; Yan, Y.; Mao, F.; Zhu, W.; Shao, Q.; Qian, H.; Xu, W., SALL4, a novel marker for human gastric carcinogenesis and metastasis. Oncogene 2014, 33 (48), 5491-5500.
(10) Yu, J.; Liang, Q. Y.; Wang, J.; Cheng, Y.; Wang, S.; Poon, T. C. W.; Go, M. Y. Y.; Tao, Q.; Chang, Z.; Sung, J. J. Y., Zinc-finger protein 331, a novel putative tumor suppressor, suppresses growth and invasiveness of gastric cancer. Oncogene 2012, 32 (3), 307-317.
(11) Cheng, Y.; Liang, P.; Geng, H.; Wang, Z.; Li, L.; Cheng, S. H.; Ying, J.; Su, X.; Ng, K. M.; Ng, M. H.; Mok, T. S.; Chan, A. T.; Tao, Q., A novel 19q13 nucleolar zinc finger protein suppresses tumor cell growth through inhibiting ribosome biogenesis and inducing apoptosis but is frequently silenced in multiple carcinomas. Mol. Cancer Res. 2012, 10 (7), 925-936.
(12) Severson, P. L.; Tokar, E. J.; Vrba, L.; Waalkes, M. P.; Futscher, B. W., Coordinate H3K9 and DNA methylation silencing of ZNFs in toxicant-induced malignant transformation. Epigenetics 2013, 8 (10), 1080-1088.
(13) Evans, P.; Liu, C., Roles of Krüpel-like factor 4 in normal homeostasis, cancer and stem cells. Acta. Biochim. Biophys. Sin. 2008, 40 (7), 554-564.
(14) Li, Y.; Wang, Y.; Zhang, C.; Yuan, W.; Wang, J.; Zhu, C.; Chen, L.; Huang, W.; Zeng, W.; Wu, X.; Liu, M., ZNF322, a novel human C2H2 Kruppel-like zinc-finger protein, regulates transcriptional activation in MAPK signaling pathways. Biochem. Biophys. Res. Commun. 2004, 325 (4), 1383-1392.
(15) Lo, F.; Chang, J.; Chang, I.; Chen, Y.; Hsu, H.; Huang, S.; Tsai, F.; Jiang, S.; Kanteti, R.; Nandi, S.; Salgia, R., .; Wang, Y., The database of chromosome imbalance regions and genes resided in lung cancer from Asian and Caucasian identified by array-comparative genomic hybridization. BMC Cancer 2012, 235 (12), 1471-2407.
(16) Jen, J.; Lin, L.-L.; Chen, H.-T.; Liao, S.-Y.; Lo, F.-Y.; Tang, Y.-A.; Hsu, H.-S.; Salgia, R.; Hsu, C.-L.; Huang, H.-C.; Juan, H.-F.; Wang, Y.-C., Oncoprotein ZNF322A transcriptionally deregulates alpha alphaalpha-adducin, cyclin D1 and p53 to promote tumor growth and metastasis in lung cancer. Oncogene 2015, (in press).
(17) Siegel, R.; Ma, J.; Zou, Z.; Jemal, A., Cancer statistics, 2014. CA. Cancer J. Clin. 2014, 64 (1), 9-29.
(18) Allemani, C.; Weir, H. K.; Carreira, H.; Harewood, R.; Spika, D.; Wang, X.; Bannon, F.; Ahn, J. V.; Johnson, C. J.; Bonaventure, A.; Marcos-Gragera, R.; Stiller, C.; Azevedo e Silva, G.; Chen, W.; Ogunbiyi, O. J.; Rachet, B.; Soeberg, M. J.; You, H.; Matsuda, T.; Bielska-Lasota, M.; Storm, H.; Tucker, T. C.; Coleman, M. P., Global surveillance of cancer survival 1995–2009: analysis of individual data for 25 676 887 patients from 279 population-based registries in 67 countries (CONCORD-2). The Lancet. 2015, 385 (9972), 977-1010.
(19) Little, A. G.; Gay, E. G.; Gaspar, L. E.; Stewart, A. K., National survey of non-small cell lung cancer in the United States: epidemiology, pathology and patterns of care. Lung Cancer 2007, 57 (3), 253-260.
(20) Brognard, J.; Clark, A.; Ni, Y.; Dennis, P., Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation. Cancer Res. 2001, 61 (10), 3986-3997.
(21) Xu, Z. H.; Hang, J. B.; Hu, J. A.; Gao, B. L., RAF1-MEK1-ERK/AKT axis may confer NSCLC cell lines resistance to erlotinib. Int. J. Clin. Exp. Pathol. 2013, 6 (8), 1493-1504.
(22) Dhillon, A.; SHagan, S.; Rath, O.; Kolch, W., MAP kinase signalling pathways in cancer. Oncogene 2007, 26 (22), 3279-3290.
(23) Itoh, N.; Semba, S.; Ito, M.; Takeda, H.; Kawata, S.; Yamakawa, M., Phosphorylation of Akt/PKB is required for suppression of cancer cell apoptosis and tumor progression in human colorectal carcinoma. Cancer 2002, 94 (12), 3127-3134.
(24) Palumbo, A. M.; Smith, S. A.; Kalcic, C. L.; Dantus, M.; Stemmer, P. M.; Reid, G. E., Tandem mass spectrometry strategies for phosphoproteome analysis. Mass Spectrom Rev. 2011, 30 (4), 600-625.
(25) Macek, B.; Mann, M.; Olsen, J. V., Global and site-specific quantitative phosphoproteomics: principles and applications. Annu. Rev. Pharmacol. Toxicol. 2009, 49, 199-221.
(26) Yoon, P. L., Mining the tumor phosphoproteome for cancer markers. Clin. Cancer Res. 2005, 11 (9), 3163-3169.
(27) Harsha, H. C.; Pandey, A., Phosphoproteomics in cancer. Mol. Oncol. 2010, 4 (6), 482-495.
(28) Olsen, J. V.; Mann, M., Status of Large-scale Analysis of Post-translational Modifications by Mass Spectrometry. Mol. Cell Proteomics 2013, 12 (12), 3444-3452.
(29) Laplante, M.; Sabatini, D. M., mTOR signaling in growth control and disease. Cell 2012, 149 (2), 274-293.
(30) Weichhart, T., Mammalian target of rapamycin: a signaling kinase for every aspect of cellular life. Methods Mol. Biol. 2012, 821, 1-14.
(31) Dowling, R. J.; Topisirovic, I.; Fonseca, B. D.; Sonenberg, N., Dissecting the role of mTOR: lessons from mTOR inhibitors. Biochim. Biophys. Acta. 2010, 1804 (3), 433-439.
(32) Dunlop, E. A.; Tee, A. R., Mammalian target of rapamycin complex 1: signalling inputs, substrates and feedback mechanisms. Cellular signalling 2009, 21 (6), 827-835.
(33) Slomovitz, B. M.; Coleman, R. L., The PI3K/AKT/mTOR pathway as a therapeutic target in endometrial cancer. Clin. Cancer. Res. 2012, 18 (21), 5856-64.
(34) Al-Batran, S. E.; Ducreux, M. O., A., mTOR as a therapeutic target in patients with gastric cancer. Int. J. Cancer 2012, 130 (3), 491-496.
(35) Feng, Z.; Zhang, H.; Levine, A. J.; Jin, S., The coordinate regulation of the p53 and mTOR pathways in cells. Proc. Natl. Acad. Sci. 2005, 102 (23), 8204-8209.
(36) Janku, F.; McConkey, D. J.; Hong, D. S.; Kurzrock, R., Autophagy as a target for anticancer therapy. Nat. Rev. Clin. Oncol. 2011, 8 (9), 528-539.
(37) Kanzawa, T.; Zhang, L.; Xiao, L.; Germano, I. M.; Kondo, Y.; Kondo, S., Arsenic trioxide induces autophagic cell death in malignant glioma cells by upregulation of mitochondrial cell death protein BNIP3. Oncogene 2005, 24 (6), 980-991.
(38) Shao, Y.; Gao, Z.; Marks, P. A.; Jiang, X., Apoptotic and autophagic cell death induced by histone deacetylase inhibitors. Proc. Natl. Acad. Sci. 2004, 101 (52), 18030-180305.
(39) Baehrecke, E. H., Autophagy: dual roles in life and death? Mol. Cell Biol. 2005, 6, 505-510.
(40) Levy, J. M.; Thorburn, A., Targeting autophagy during cancer therapy to improve clinical outcomes. Pharmacol. Ther. 2011, 131 (1), 130-141.
(41) Sui, X.; Kong, N.; Ye, L.; Han, W.; Zhou, J.; Zhang, Q.; He, C.; Pan, H., p38 and JNK MAPK pathways control the balance of apoptosis and autophagy in response to chemotherapeutic agents. Cancer Lett. 2014, 344 (2), 174-179.
(42) Rappsilber, J.; Mann, M.; Ishihama, Y., Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2007, 2 (8), 1896-1906.
(43) Hennrich, M. L.; Mohammed, S.; Altelaar, A. F.; Heck, A. J., Dimethyl isotope labeling assisted de novo peptide sequencing. J. Am. Soc. Mass Spectrom 2010, 21 (12), 1957-1965.
(44) Sugiyama, N.; Masuda, T.; Shinoda, K.; Nakamura, A.; Tomita, M.; Y., I., Phosphopeptide Enrichment by Aliphatic Hydroxy Acid-modified Metal Oxide Chromatography for Nano-LC-MS/MS in Proteomics Applications. MCP 2007, 6 (6), 1103-1109.
(45) Cox, J.; Neuhauser, N.; Michalski, A.; Scheltema, R. A.; Olsen, J. V.; Mann, M., Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 2011, 10 (4), 1794-1805.
(46) Cox, J.; Mann, M., MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 2008, 26 (12), 1367-1372.
(47) Schwartz, D.; Gygi, S. P., An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets. Nat. Biotechnol. 2005, 23 (11), 1391-1398.
(48) Hu, J.; Rho, H.; Newman, R.; Zhang, J.; Zhu, H.; Qian, J., PhosphoNetworks: a database for human phosphorylation networks. Bioinformatics 2014, 30 (1), 141-142.
(49) Li, C.; Liu, H.; Sun, Y.; Wang, H.; Guo, F.; Rao, S.; Deng, J.; Zhang, Y.; Miao, Y.; Guo, C.; Meng, J.; Chen, X.; Li, L.; Li, D.; Xu, H.; Wang, H.; Li, B.; Jiang, C., PAMAM nanoparticles promote acute lung injury by inducing autophagic cell death through the Akt-TSC2-mTOR signaling pathway. J. Mol. Cell Biol. 2009, 1 (1), 37-45.
(50) Hung, J. Y.; Hsu, Y. L.; Li, C. T.; Ko, Y. C.; Ni, W. C.; Huang, M. S.; Kuo, P. L., 6-Shogaol, an active constituent of dietary ginger, induces autophagy by inhibiting the AKT/mTOR pathway in human non-small cell lung cancer A549 cells. J. Agric. Food Chem. 2009, 57 (20), 9809-9816.
(51) Li, P.; Shi, J.; He, Q.; Hu, Q.; Wang, Y. Y.; Zhang, L. J.; Chan, W. T.; Chen, W. X., Streptococcus pneumoniae induces autophagy through the inhibition of the PI3K-I/Akt/mTOR pathway and ROS hypergeneration in A549 cells. PloS one 2015, 10 (3), e0122753.
(52) Gridelli, C.; Maione, P.; Rossi, A., The potential role of mTOR inhibitors in non-small cell lung cancer. The oncologist 2008, 13 (2), 139-147.
(53) Liu, P.; Cheng, H.; Roberts, T. M.; Zhao, J. J., Targeting the phosphoinositide 3-kinase pathway in cancer. Nat. Rev. Drug Discov. 2009, 8 (8), 627-644.
(54) Inoki, K.; Li, Y.; Xu, T.; Guan, K. L., Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev. 2003, 17 (15), 1829-1834.
(55) Jung, C. H.; Ro, S. H.; Cao, J.; Otto, N. M.; Kim, D. H., mTOR regulation of autophagy. FEBS Lett. 2010, 584 (7), 1287-1295.
(56) Yang, Z. J.; Chee, C. E.; Huang, S.; Sinicrope, F. A., The role of autophagy in cancer: therapeutic implications. Mol. Cancer Ther. 2011, 10 (9), 1533-1541.


QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top