博碩士論文 100233601 詳細資訊



以作者查詢圖書館館藏 、以作者查詢臺灣博碩士 、以作者查詢全國書目 、勘誤回報 、線上人數：8 、訪客IP：13.59.61.119
姓名 桑瑞蒙(FX Reymond Sutandy)  查詢紙本館藏   畢業系所 系統生物與生物資訊研究所
論文名稱 異質性核醣核酸蛋白K (hnRNP K) 抑制成熟miRNA-122轉錄後調控機制之研究
(Heterogeneous ribonucleoprotein K (hnRNP K) inhibits the post-transcriptional regulation of mature miRNA-122)
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摘要(中) MiRNA可募集許多RNA結合蛋白(RNA binding protein, RBP)，為一個相當有趣的生物現象。儘管目前學者對於RBP調控未成熟miRNA的生合成已有相當了解，而彼等對於RBP調控成熟miRNA等生物功能則未有指出。有鑑於此．本研究利用人類蛋白質體晶片(包含約17,000個蛋白質)與miRNA-122為模式，成功篩選40個miRNA-122結合蛋白。進一步對此miRNA-122結合蛋白之生物形象進行分析，共定義4個成熟的miRNA-122結合蛋白。研究利用客製建構之miRNA-122標的序列DNA晶片，並測試此4種成熟miRNA-122結合蛋白對其干擾miRNA與其標的DNA雜和顯示，hnRNPK可結合miRNA-122 (70%)或miRNA-122標的序列(30%)進而抑制miRNA-122與miRNA-122標的DNA序列之雜和。且彼等雜和之抑制可隨hnRNP K含量呈劑量反應，充分顯示hnRNP K的專一性。體內研究利用AML12細胞株並大量表現hnRNP K顯示，hnRNP K可降低miR-122對於Aldolase A的抑制作用，且不影響AML12內源miR-122的生合成。綜觀，本研究以成熟miR122為模式發現，hnRNP K可結合miR122並抑制其轉錄後之調控。本研究乃提供一新穎之miRNA調控機制，其對於miRNA領域具有重要影響。彼等研究成果可進一步用於miRNA相關疾病之治療。
摘要(英) MiRNA regulation is an intriguing biological process that recruits several RBPs. Although the regulation of miRNA biosynthesis has been thoroughly studied, a direct regulation through binding to mature miRNA has not been reported. Using a human proteome chip which consist of ~17.000 unique human proteins and miR-122 as a model, we identified 40 miR-122-binding proteins. After further screening by their biological profiles, four mature miR-122-binding proteins were identified. We established the DNA chip containing miR-122 target sequence to test the capability of our identified proteins to interfere the hybridization between miR-122 and its target sequence. Among the four miR-122-binding proteins, only heterogenous ribonucleoprotein K (hnRNP K) showed the most significant ability (70%) to inhibit the hybridization through binding to miR-122, while mild inhibition (30%) through binding to miR-122-targetting sequence was also observed. The hybridization inhibition was proportionally related to the amount of hnRNP K in a dose response manner, indicating the specificity of this inhibition. In vivo study, overexpression of hnRNP K in AML12 cell line significantly decreased the inhibition of miR-122 toward its target mRNA of Aldolase A without affecting the biosynthesis of miR122 itself. In summary, we hypothesized that hnRNP K exhibited a novel regulation toward miR122 by directly bind to it and inhibit its function of posttranscriptional regulation. Although further study was necessary, our finding provided a new direction of miRNA regulation study which might be important for development of miRNA-related diseases therapies.
關鍵字(中) ★ miRNA-122
★ 異質性核醣核酸蛋白K		 關鍵字(英)
論文目次 Table of Contents
中文摘要 i
ABSTRACT ii
ABBREVIATIONS iii
ACKNOWLEDGMENTS iv
Table of Contents v
List of Figures vii
List of Tables viii
Chapter 1 Overview of Protein Microarrays 1
1.1 History 1
1.2 Three Categories 2
Analytical Protein Microarrays 2
Functional Protein Microarrays 3
Reverse-Phase Protein Microarrays 4
1.3 Fabrication of Protein Microarrays 5
Protein Production 6
Surface Chemistry 7
1.4 Detection 7
1.5 Applications in Basic Research 9
Development of new assays 9
Detection of Protein-Binding Properties 10
Profiling monoclonal antibody specificity 15
Protein posttranslational modifications 15
1.6 Applications in Clinical Research 19
Host-microbe interactions 19
Biomarker identification 21
1.7 Future Prospects 23
Chapter 2 High throughput platform to exploring RNA proteome interactions 28
2.1 Introduction 28
2.2 Standard methods for detecting RBP-RNA interaction 29
2.3 Pull down of RBP-RNA complex 30
RNA vs RBP bait format 31
Tag-fusion strategy 32
2.4 Protein microarrays platform for RBP-RNA interaction study 33
Protein microarrays platform for RBP-RNA study 33
RNA labeling strategies and detections 34
2.5 Applications of high throughput screening of RBP-RNA interactions 35
Profiling RNA expression 35
Studying RBP-related functions and regulations 36
Diseases development studies 37
2.6 Future prospects 38
Chapter 3 Heterogeneous ribonucleoprotein K (hnRNP K) inhibits the post-transcriptional regulation of mature miRNA-122 42
3.1 Introduction 42
3.2 Materials and Methods 44
Human proteome chip assay 44
DNA chip fabrication 45
DNA chip assays for hybridization interruption of miR-122 and its target sequence 45
DNA chip assays for binding between proteins and miR-122 target sequence 46
Electrophoretic mobility shift assay (EMSA) 46
Dose response of hnRNP K for the hybridization inhibition between miR-122 and its target sequence 46
Cell line transfections 47
Western Blot 47
Quantitative reverse transcription-PCR (qRT-PCR) 48
3.3 Result 49
Identification of human mature miR-122-binding proteins 49
In vitro test for the capability of identified proteins to inhibit mature miR-122 binding to the target sequence 50
Validation of hnRNP K binding to mature miR122 52
hnRNP K inhibited miR122 activity toward its target in vivo 53
3.4 Discussion and Conclusion 54
REFERENCES 65
APPENDIXES 84
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指導教授 陳健生(Chien-Sheng Chen) 審核日期 2013-7-30
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