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研究生:鍾雯玥
研究生(外文):Wen Ye Tjong
論文名稱:CD97/ADGRE5興EMR2/ADGRE2中RGD Motif 對於人類纖維肉瘤細胞HT1080的細胞貼附, 細胞存活, 及癌細胞血管生成功能之影響
論文名稱(外文):The RGD motif of CD97/ADGRE5 and EMR2/ADGRE2 is involved in cell adhesion, cell viability, and tumor angiogenesis of human HT-1080 fibrosarcoma cell line
指導教授:林錫賢
指導教授(外文):H. H. Lin
學位類別:博士
校院名稱:長庚大學
系所名稱:生物醫學研究所
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:2019
畢業學年度:108
語文別:英文
論文頁數:109
中文關鍵詞:CD97EMR2細胞貼附细胞聚集N-cadherinMMP9凋亡血管生成
外文關鍵詞:CD97EMR2cell adhesioncell aggregationN-cadherinMMP9ApoptosisAngiogenesis
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ACKNOWLEDGEMENT iii
ABSTRACT iv
TABLE OF CONTENTS vi
LIST OF FIGURES ix
LIST OF TABLES xi
CHAPTER I INTRODUCTION 1
A. Adhesion GPCR and the EGF-TM7 family 1
B. CD97/ADGRE5 2
C. EMR2/ADGRE2 4
D. Arginine-Glycine-Aspartic (RGD) Motif 6
F. The relationship between MMP9, N-cadherin, and Angiogenesis 7
CHAPTER II AIMS OF THE STUDY 10
CHAPTER III MATERIALS AND METHODS 11
A. Reagents and antibodies. 11
B. Vector construction 12
C. Retroviral infection and selection of stable HT1080 cell lines 13
D. Western blot and flow cytometry analysis 13
E. Cell aggregation assay 14
F. Cell adhesion assay 15
G. Cell viability assay 16
H. Apoptosis assay 17
I. MMP zymography 17
J. RT-PCR and quantitative real-time PCR analysis 18
K. Small interfering rna transfection 18
L. ELISA assay to detect hVEGF, PIGF, and bFGF protein 19
M. In vitro Matrigel tube formation assay 19
N. HUVEC proliferation assay 20
O. Migration and invasion assay 20
P. Chorioallantoic membrane (CAM) assay 21
Q. Statistical analysis 21
CHAPTER IV RESULTS 23
A. Generation of stable HT1080 cell lines expressing recombinant CD97 and
EMR2 receptors 23
B. The RGD sequence on the stalk region of CD97 is involved in promoting cell
adhesion by upregulating the expression of integrin αvβ5 and α2β1 24
C. The RGD sequence on the stalk region of CD97 and EMR2 is involved in the
inhibition of cell apoptosis 25
D. The RGD sequence on the stalk region of EMR2 increases N-cadherin
expression then stimulates HT1080 homotypic cell aggregation . 27
E. N-cadherin overexpression increases cell survival of CD97 and EMR2 28
F. HT1080 cells expressing CD97 and EMR2-RGD isoforms secreted upregulated MMP9 and promoted in vitro angiogenesis 30
G. Conditioned medium of HT1080 cells expressing CD97 and EMR2-RGD
isoforms modulated HUVEC cell proliferation, migration, and invasion 31
H. MMP9 is the major factor in the HT1080 cell CM modulating in vitro and in ovo
angiogenesis and HUVEC cell proliferation, migration, and invasion 32
I. The expression of pro-angiogenic factors is modulated by MMP9 in conditioned
medium of HT1080 cells expressing CD97 and EMR2-RGD isoforms. 34
J. Up-regulated N-cadherin by CD97 stimulates the synthesis of MMP9 . 35
CHAPTER V DISCUSSION 36
A. The RGD motif is involved in CD97/ADGRE5-promoted cell adhesion and
viability of HT1080 cells 36
B. The role of the RGD motif in CD97/ADGRE5- and EMR2/ADGRE2-modulated
tumor angiogenesis . 41
REFERENCES 46
FIGURES 56
TABLES 82
SUPPLEMENTARY FIGURES 88

LIST OF FIGURES
Figure 1. Different variants of (A) CD97 and (B) EMR2 based on EGF-like isoforms.
57
Figure 2. Generation of HT1080 cells stably expressing genetically engineered
CD97 and EMR2 proteins. . 58
Figure 3. Enhanced HT1080 cell adhesion induced by CD97 and EMR2 is
dependent in part on the RGD motif 61
Figure 4. The RGD motif of CD97 promotes cell viability. 63
Figure 5. The RGD motif of CD97 inhibits extrinsically-induced apoptosis. 64
Figure 6. The RGD motif of EMR2 inhibits intrinsically-induced apoptosis. 65
Figure 7. The RGD motif of EMR2 promotes N-cadherin expression resulting in
enhanced HT1080 cell aggregation. 67
Figure 8. Enhanced cell aggregation promoted by up-regulated N-cadherin inhibits
HT1080 cell apoptosis. 69
Figure 9. Conditioned medium of HT1080 cells expressing CD97 and EMR2-RGD
isoforms contained up-regulated levels of MMP9 and promoted in vitro
angiogenesis 71
Figure 10. The effect of conditioned medium of distinct HT1080 stable cells on the
proliferation, migration, and invasion of HUVEC cells. 73
Figure 11. MMP9 is the major factor in the HT1080 cell CM modulating in vitro
HUVEC cell angiogenesis as well as cell proliferation, migration, and
invasion. 75
Figure 12. Chorioallantoic membrane (CAM) assay for in ovo angiogenesis 78
Figure 13. The expression of pro-angiogenic factors in HT1080 stable cells is
modulated by MMP9 79
Figure 14. The role of N-cadherin in the regulation of MMP9 expression 81

Suppplementary Fig 1. Flow cytometry analysis of the expression of selective
integrin molecules on different HT1080 stable cells as indicated 89
Supplementary Fig 2. The levels of active β3 integrin were examined by flow
cytometry analysis after stable cells were starved overnight. . 90
Suppplementary Fig 3. The flow cytometry analysis of cell apoptosis of indicated
stable HT1080 cells. 91
Supplementary Fig 4. N-cadherin-promoted cell aggregation inhibits HT1080 cell
apoptosis. 92
Supplementary Fig 5. Conditioned medium of HT1080 cells expressing CD97 and
EMR2-RGD isoforms contained up-regulated levels of MMP9 and
promoted in vitro angiogenesis. . 95
Supplementary Fig. 6. The gene expression profile across all tumor samples and
paired normal tissues is determined using GEPIA website . 96
Supplementary Fig. 7. Gene correlation analysis was determined by GEPIA
website. . 97

LIST OF TABLES
Table 1. Primers used in the quantitative real-time PCR analysis 83
Table 2. ABBREVIATIONS 84
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