臺灣博碩士論文加值系統

Signal peptide-CUB-EGF-like domain-containing protein 3 (SCUBE3) 是一個經過糖修飾的分泌型蛋白，在肺癌的腫瘤組織中已被發現會大量表現，也與肺癌細胞的轉移能力有相關。在先前的研究已指出SCUBE3扮演與TGF-β相似的重要角色，藉由TGF-β receptors-Smad2/3 pathway來調控腫瘤發展。因此，能找到SCUBE3專一性的抑制劑對於肺癌的治療應用相當具有潛力。適體(Aptamers)為短片段的DNA或是RNA，對於其標定的分子可以形成高度專一性二級結構。在這次的研究中，我們用systematic evolution of ligands by exponential enrichment (SELEX)的方式針對SCUBE3的重組蛋白來進行單股DNA 適體篩選。目前，我們已經篩選出SCUBE3-specific aptamers，S3A1(Dissociation constant : 18nM) 。且跟Control cell 相比，S3-A1傾向與 SCUBE3-overexpressed cells做結合。在TGF-β receptors-Smad2/3 signaling，S3-A1對lung cancer cell似乎會增加磷酸化Smad2的表現，所以可能暗示著 S3-A1會促進活化SCUBE3。藉由Wound-healing Assay我們發現，S3-A1促進SCUBE3-induced cancer cells 遷移。所以接下來，我們將進一步調查S3-A1對cancer cells 和vascular endothelial cells由SCUBE3所調控影響的生物功能。因此，目前可以了解S3-A1直接 與SCUBE3結合，且可能會影響SCUBE3/TGFβR/Smad2 signaling 並調控癌症的發展之外，也在體外細胞實驗中發現S3-A1能夠阻斷內皮細胞血管生成。

Signal peptide-CUB-EGF-like domain-containing protein 3 (SCUBE3), a secreted glycoprotein, has been found to be overexpressed in lung cancer tumor tissues and correlated with the invasive ability of lung cancer cells. Previous study has showed that SCUBE3 may have a critical role with TGF-β, and control tumorigenesis via the TGF-β receptors-Smad2/3 pathway. Here, our hypothesis is SCUBE3-specific inhibitors may have potential therapeutic applications for lung cancer. Aptamers are short DNA or RNA fragments that can form complex secondary structures to have specific ability for its target molecule. In this study, we used recombinant SCUBE3 proteins to screen single-stranded DNA aptamers by systematic evolution of ligands by exponential enrichment, SELEX, and identified a SCUBE3-specific aptamer, S3-A1. We found that biotinylated S3-A1 specifically binds to SCUBE3 proteins with an around 18nM dissociation constant. S3-A1 also preferentially binds to SCUBE3-overexpred cells compared with control cells. Treatment of S3-A1 on lung cancer cells seemed to increase phosphorylation of Smad2 implying that S3-A1 might promote SCUBE in TGF-β receptors-Smad2/3 pathway activation. By wound-healing assay, we found that S3-A1 promoted SCUBE3-induced cancer cells migration. Next, we will further investigate the influences of S3-A1 on SCUBE3-mediated biological functions of cancer cells and vascular endothelial cells. In conclusion, S3-A1 may affect SCUBE3/TGFβR/Smad2 signaling by directly binding SCUBE3 and regulate cancer progression and it also inhibits in vitro tube formation of vascular endothelial cells.

摘要 I
Abstract II
ACKNOWLEDGEMENT III
CONTENTS V
ABBREVIATION VIII
INTRODUCTION 1
1. Aptamer 1
Origin of Aptamer 1
Superiority of Aptamer 1
2. SCUBE3 3
Signal peptide-CUB-EGF-like domain-containing protein 3 3
Relevance of SCUBE3 in Cancers 4
RATIONALE AND SPECIFIC AIMS 6
MATERIALS AND METHODS 7
Cell Lines and Culture 7
Nitrocellulose Membrane Filtration-Based SELEX 7
Alignment and Structure Prediction 8
Surface Plasmon Resonance – Biacore3000 8
Magnetic Beads Pull-down Assay 9
Immunofluorescence 9
Cell Morphology 9
Cell Proliferation – WST-1 assay 10
Wound Healing Assay 10
Tube Formationy 10
Western Blot Analysis 11
Statistical Analysis 12
RESULTS 13
DNA aptamer against SCUBE3 13
Specificity of S3-A1 for SCUBE3 13
Functions of S3-A1 for lung cancer cells 14
S3-A1 enhances SCUBE3/TGF-β receptor signaling 15
Function of SCUBE3 for Angiogenesis 15
Function of S3-A1 for Angiogenesis 16
DISSCUSION 17
CONCLUSION 20
REFERRENCES 21
TABLE 26
Table. 1 Preparation for SELEX buffer 26
Table. 2 Preparation for PCR 27
Primer used in PCR (Invitrogen) 27
PCR Reagents 27
PCR Program 27
Table. 3 Aptamers used in Experiments 28
FIGURE 29
Figure 1. Flowchart of Membrane SELEX. 29
Figure 2. Isolation and Identification of S3-A1. 30
Figure 3. Specificity of S3-A1 to SCUBE3. 32
Figure 4. S3-A1 increases migration of cancer cells. 33
Figure 5. S3-A1 does not affect proliferation of cancer cells. 34
Figure 6. S3-A1 induces epithelial–mesenchymal transition (EMT) of lung cancer cells. 35
Figure 7. S3-A1 enhances SCUBE3/TGF-β receptor signaling. 36
Figure 8. SCUBE3 regulates HUVECs tube formation. 37
Figure 9. S3-A1 inhibits HUVECs tube formation through blockading pAkt. 39
APPENDIX 40
Appendix 1. Antibodies 40
Appendix 2. Reagents 41
Appendix 3. Equipment 42

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