Title Page
ABSTRACT
Contents
LIST OF ABBREVIATIONS 17
1. INTRODUCTION 18
1.1. Prostate cancer characteristics 18
1.1.1. Androgen receptor (AR) signaling and androgen deprivation therapy (ADT) 19
1.1.2. Molecular features of prostate cancer aggressiveness 27
1.2. Differential expression of integrin molecules in ARPC and their regulatory mechanisms 41
1.2.1. Types of integrins and their role in prostate cancer progression 41
1.2.2. Regulation of integrin expression 47
1.3. Possible role of platelets in prostate cancer progression 59
2. MATERIALS AND METHODS 72
2.1. Cell line and cell culture 72
2.2. Transcriptome analysis 72
2.3. miRNA expression analysis 73
2.4. Protein extraction and immunoblotting 74
2.5. Transfection with miRNA/siRNA 75
2.6. Cell viability and proliferation assay 76
2.7. qRT-PCR 77
2.8. Sphere formation assay 78
2.9. Measurement of prostate cancer stem cell (PCSC) population 79
2.10. Fluorescence microscopy 80
2.11. miRNA target prediction and 3'UTR target clones and dual luminescence assay 80
2.12. Chromatin Immuno-precipitation (ChIP) Assay 81
2.13. Prostate cancer cell co-culture with MEG-01 cells 82
2.14. Determination of MEG-01-derived microparticle migration and viable cancer cell number 82
2.15. Cytokine array 83
2.16. Chemokine ELISA 84
2.17. Cancer cell invasion assay 84
2.18. Cancer stem cell transcription factor (TF) activation profiling plate array 85
2.19. Tumor xenograft on Chick Chorioallantoic membrane (CAM) 85
2.20. Statistical analysis 86
3. RESULTS 87
3.1. Differentially expressed integrin genes, ITGA3 and ITGB4, in ARPC cell lines maintain prostate cancer survival and PCSCs population 87
3.2. Differentially expressed miRNAs regulate androgen refractory genes in ARPC cell lines 100
3.3. miR-200c-3p suppresses integrin α3/β4 expression in a dual mode 116
3.4. miR-200c-3p mimic inhibits tumor growth and metastasis of implanted PC-3 cells in a xenografted tumor model. 126
3.5. Endogenous IL-8 level is significantly high and correlates with upregulated invasiveness in ARPC compared to LNCaP 134
3.6. Platelet precursor cells, MEG-01, secrete IL-8 in co-culture with prostate cancer cells 140
3.7. Co-culture with MEG-01 cells upregulates expression of integrin α3 and β4 and prostate cancer progression 145
3.8. IL-8-CXCR2 signaling is involved in MEG-01 cell-mediated increase in invasion and metastasis of prostate cancer cells 152
4. DISCUSSION 159
BIBLIOGRAPHY 170
요약 191
Table 1. Classification of MMPs and expression in prostate cancer 32
Table 2. PCSCs markers and their localization 38
Table 3. Different integrins and their Ligand-binding specificities 42
Table 4. miRNAs expression in various cancers 54
Table 5. miRNAs in prostate cancer 57
Table 6. Secretome of an activated platelets 64
Table 7. Pro-tumorigenic roles of various chemokines 68
Table 8. Primer sequences for qRT-PCR 77
Table 9. The list of top 30 genes with significantly different expressions in PC-3 and DU145 cells compared to LNCaP cells (p〈0.05) 97
Table 10. Relative integrin gene expressions with significant difference in PC-3 and DU145 cells compared to LNCaP cells 99
Table 11. Differentially up- and down-regulated miRNAs in DU145 and PC-3 cells compared to LNCaP cells (p〈0.05) 109
Table 12. Differentially expressed miRNAs and their predicted target genes selected from DEGs commonly up- and down-regulated in PC-3... 111
Table 13. IC50 of various compounds on viability of LNCaP and PC-3 cells 118
Fig. 1. Integrin α3 and β4, ZBTB16 (PLZF) and AR are differentially up- and down-regulated in ARPC compared to LNCaP. 92
Fig. 2. Silencing integrin α3 and β4 inhibits ARPC viability and CSC population in ARPC, without altering PLZF and AR expression level. 95
Fig. 3. miR-200c-3p binds to and inhibits expression of ITGA3/B4 mRNA. 108
Fig. 4. miR-200c-3p inhibits expression of Integrin α3 and β4 but increases PLZF, AR expression in prostate cancer cells. 115
Fig. 5. miR-200c-3p-induced PLZF inhibits Integrin α3 and β4 expression which increases sensitivity to Enzalutamide (anti-androgen drug). 122
Fig. 6. Additive effect of miR-200c-3p inhibitor and PLZF siRNA on prostate cancer stem cell increase. 125
Fig. 7. miR-200c-3p mimic restores androgen sensitivity and inhibits tumor growth and metastasis of implanted PC-3 cells in a xenografted... 132
Fig. 8. Endogenous CXCL8/IL-8 level is significantly high and correlates with upregulated invasiveness of ARPC compared to LNCaP. 139
Fig. 9. Platelet pre-cursor cells, MEG-01, secrete IL-8 in co-culture with prostate cancer cells. 142
Fig. 10. MEG-01 cells secrete IL-8 in cultured media 144
Fig. 11. Co-culture with MEG-01 cells upregulates Integrin α3 and β4, MMP-2, EMT markers, stem cell characteristics and invasiveness of... 151
Fig. 12. IL-8-CXCR2 signaling is involved in MEG-01 cells mediated increase in invasion and metastasis of prostate cancer cells. 155
Fig. 13. CXCR2 signaling is strongly involved in increased metastasis of ARPC in a xenografted tumour model. 157
Diagram 1. Androgen receptor with its domains 20
Diagram 2. AR signaling 22
Diagram 3. Steps of metastasis 29
Diagram 4. miRNA synthesis and functions 49
Diagram 5. TME components 60
Diagram 6. Electronic microscopic image of a platelet exhibiting cytosolic and granule ultrastructures 63