臺灣博碩士論文加值系統

鋅指蛋白322A (ZNF322A)屬於C2H2型鋅指蛋白家族並擁有轉錄因子的功能。近年的研究指出，在肺癌病患檢體中，ZNF322A有過度表現的現象，可能為肺癌的致癌基因之一，然而，ZNF322A調控的網路尚未被徹底解析。在本研究中，我們結合了定量蛋白質體學及磷酸化蛋白質體學的方法，分析ZNF322A在人類肺腺癌細胞株A549的調控網路，利用羥基酸修正金屬氧化物層析(hydroxy acid-modified metal oxide chromatography, HAMMOC)、相對定量同位素標定(isobaric tag for relative and absolute quantitation, iTRAQ)及液相層析與串聯式質譜儀(LC-MS/MS)的技術，我們發現2754個磷酸化位置對應到1822個磷酸肽及976個磷酸蛋白上，其中共有138個磷酸化位置受ZNF322A調控。比對先前的蛋白質體資料並利用功能富集、生物網路和磷酸化基序列分析等生物資訊學的方法，我們發現ZNF322A可能參與RNA加工及細胞週期等調控，且熱休克蛋白27 (HSP27)的Ser82磷酸化位置有顯著的表現量上升，並與轉錄起始因子eIF2α一同參與啟動未摺疊蛋白反應(unfolded protein response)，另外，IRS1 Ser1101磷酸化位置的表現量上升可能參與調控葡萄糖的運送，結果指出HSP27與IRS1為ZNF322A下游的調控磷酸化蛋白。本研究對於ZNF322A在人類肺癌細胞中的分子調控機制有更深入的了解，期望能對肺癌的治療發展有所貢獻。

ZNF322A is a transcription factor that belongs to C2H2-type zinc finger protein family. Recent study revealed that ZNF322A is a potential oncogene in lung cancer patients. However, the network regulated by ZNF322A was still unknown. In this study, we integrated quantitative phosphoproteomic and proteomic analyses for ZNF322A-silencing lung cancer A549 cells. Hydroxy acid-modified metal oxide chromatography (HAMMOC) phosphopeptide enrichment method, and isobaric tag for relative and absolute quantitation (iTRAQ) coupled with LC-MS/MS were applied. We identified 2754 phosphosites corresponding to 1822 phosphopeptides and 976 phosphoproteins, among these, 53 phosphosites were upregulated and 85 were downregulated in siZNF322A treated cells. Quantitative iTRAQ LC-MS/MS proteomics reveals the proteome profile in ZNF322A-silencing A549 cells, and 1108 proteins were identified. By bioinformatics approaches including functional enrichment, network and phosphorylation motif analyses, the results indicated that ZNF322A might regulate several biological processes, such as RNA processing and cell cycle. Phosphorylation of HSP27 Ser82 and IRS1 Ser1101 were significantly upregulated upon ZNF322A-silencing and participate in the regulation of unfolded protein response and glucose uptake. Our study provides a new insight for further investigation of protein phosphorylation status in ZNF322A-mediated lung cancer cells.

口試委員會審定書 i
誌謝 ii
中文摘要 iii
Abstract iv
Abbreviation vi
Contents viii
List of Figures xi
List of Tables xiii
Chapter 1 Introduction 1
1.1 Zinc finger proteins 1
1.1.1 Introduction 1
1.1.2 ZNF322A 2
1.2 Lung cancer 3
1.3 Phosphoproteomics 4
1.3.1 Introduction 4
1.3.2 Phosphoproteomics in cancer 5
1.4 Endoplasmic reticulum stress and unfolded protein response 6
1.4.1 Introduction 6
1.4.2 PERK–eIF2α–ATF4 signaling pathway 7
1.5 Motivation 8
Chapter 2 Materials and Methods 9
2.1 Cell culture 9
2.2 Cell transfection 9
2.3 RNA extraction and cDNA synthesis 9
2.3 Real-time quantitative RT-PCR (qRT-PCR) assays 10
2.4 Stage tip preparation 10
2.5 Sample preparation and extraction 11
2.6 Sample preparation for phosphoproteome profiling 12
2.6.1 Dimethyl labeling of peptides 12
2.6.2 Desalting with SDB-XC StageTips 13
2.6.3 Phosphopeptide enrichment with HAMMOC and fractionation 13
2.7 NanoLC–MS/MS analysis 14
2.8 Data analysis for phosphoproteomics 15
2.9 Comparison of proteome and phosphoproteome profiles 16
2.10 Functional enrichment analysis 16
2.11 Kinase activity map analysis and protein-protein interaction network construction 17
2.12 Western blot analysis 17
2.13 Glucose uptake assay 18
Chapter 3 Results 20
3.1 ZNF322A knockdown by small interfering RNA in A549 20
3.2 Quantitative phosphoproteomic analysis of ZNF322A-silencing lung cancer A549 cells 20
3.3 Integrative analysis of ZNF322A-regulatory proteome and phosphoproteome in lung cancer A549 cells 23
3.4 Biological processes and pathways regulated by ZNF322A 24
3.5 Kinase activity map and protein-protein interaction networks analyses 25
3.6 Heat shock protein 27 phosphorylation and unfolded protein response 26
3.7 IRS1–AKT pathway attenuation and reduction of glucose uptake 27
Chapter 4 Discussion 29
Chapter 5 Conclusions 37
References 38
Figures 51
Tables 75
Appendix 102

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