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Introduction 1
I. The clinical significance of esophageal cancer in Taiwan 1
(a) The unique clinical and epidemiological characteristics of esophageal cancer in Taiwan 1
(b) Current diagnostic tools and therapeutic approaches of esophageal cancer 2
II. The molecular alterations and molecular diagnostic markers in ESCC 3
(a) Genetic alterations and known biomarker candidate genes in ESCC cancers 3
(b) Epigenetic alterations in ESCC cancers 4
(c) Known DNA hypermethylation candidate biomarkers in ESCC 6
III. Overview of SOX family and SOX17 gene 7
(a) The characteristics of SOX family 7
(b) Overview of SOX17 gene 8
(c) The role of SOX17 in Wnt signaling pathway and its tumor suppressive mechanism 9
(d) Molecular alterations of SOX17 in cancers 10
Study Basis and Specific Aims 12
Materials and Methods 13
I. Clinical samples of ESCC patients 13
II. Cell lines and culture 13
III. Genomic DNA extraction and sodium bisulfite conversion 14
IV. The Genome-wide methylation analysis platforms 14
V. Pyrosequencing assay 15
VI. RNA extraction and quantitative reverse transcription-PCR 16
VII. Immunohistochemistry (IHC) staining 16
VIII. 5-aza-2’-deoxycitidine (5-aza-dC) treatment 17
IX. Expression vector constructs and transfection 17
X. Western blot analysis 18
XI. MTT assay 19
XII. Colony formation assay 19
XIII. Wound healing assay 19
XIV. Transwell migration and invasion assays 20
XV. Expression array analysis 20
XVI. Anti-tumor growth study in animal model 21
XVII. Biological software analysis 22
XVIII. Statistical analysis 22
Results 23
I. DNA methylation biomarkers study 23
(a) Genome-wide methylation study by Illumina GoldenGate technique 23
(b) Identification of 28 prognosis related probes by Prediction Analysis for Microarrays (PAM) 24
(c) Identification of 10 cancer-related survivability genes by Level-based-mining method 25
(d) Validation of candidate methylated probes with prognostic potential by Kaplan-Meier survival curves 25
(e) Concordance between methylation level measured by Illumina GoldenGate technique and pyrosequencing assay 26
(f) Identification of candidate tumor suppressor gene SOX17 with prognostic potential 26
II. SXO17 study in clinical models 27
(a) SOX17 gene is frequently hypermethylated in ESCC patients 27
(b) SOX17 mRNA and protein are frequently low expression in ESCC patients 28
(c) A significant inverse correlation between DNA methylation and mRNA expression, as well as protein expression 29
(d) Patients with SOX17 DNA hypermethylation or low SOX17 protein expression show poor prognosis 29
III. SXO17 study in cell model 30
(a) SOX17 is hypermethylated and SOX17 mRNA and protein are down-regulated in ESCC cell lines 30
(b) Restoration of SOX17 expression through treatment with demethylation reagent 5-aza-dC 30
(c) SOX17 overexpression decreases colony formation ability of ESCC cells 31
(d) SOX17 overexpression decreases cancer cell migration and invasion 31
(e) SOX17 overexpression decreases expression of downstream genes in Wnt signaling pathway 32
(f) SOX17 overexpression decreases mRNA expression of genes other than Wnt signaling pathway 33
IV. SXO17 study in animal model 34
SOX17 overexpression decreases tumor growth in vivo 34
Discussion 35
References 43
Tables 51
Figures 63
Appendix tables and figures 89
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