Title Page
Contents
ABSTRACT 11
Chapter 1. Literature Review 13
1.1. Introduction 13
1.2. Diagnosis of Bladder Cancer 14
1.2.1. Voided Urine 15
1.3. Treatment of Bladder Cancer 18
1.3.1. Non-muscle-invasive Bladder cancer 19
1.3.2. Muscle-invasive Bladder Cancer 20
Chapter 2. Development of a technique for diagnosis and screening of superficial bladder cancer by cell-pellet DNA from urine sample 22
2.1. Introduction 22
2.2. Materials and Methods 25
2.2.1. Study Population 25
2.2.2. Urine Collection and Sample Processing 25
2.2.3. Targeted PCR and Sanger Sequencing 26
2.2.4. Selection of BCa Driver Genes 27
2.2.5. Whole Exome Sequencing 27
2.2.6. Whole Genome Sequencing 28
2.2.7. Bioinformatics Pipeline 28
2.2.8. Statistical Analysis 29
2.3. Results 31
2.3.1. Clinicopathologic features of patients and DNA extraction from the urine 31
2.3.2. Mutation frequency of coding genes from tissues and urinary cell pellets of BCa 36
2.3.3. Frequency of non-coding mutations in tissues and urinary cell pellets of BCa 37
2.3.4. Overlapping variant analysis of tissues and urinary cell pellets of BCa 40
2.3.5. Somatic alterations in signaling pathways from patient samples with BCa and paired urine cell pellets 45
2.3.6. Identification of specific mutations in BCa driver genes in cpDNA 48
2.3.7. Estimation and comparison of TMB 50
2.4. Discussion 52
Chapter 3. Anti-oncogenic effects of dutasteride, a dual 5-alpha reductase inhibitor and a drug for benign prostate hyperplasia, in bladder cancer 55
3.1. Introduction 55
3.2. Materials and Methods 57
3.2.1. Cell lines and treatments 57
3.2.2. Reverse Transcription-Polymerase Chain Reaction and Quantitative real-time Polymerase Chain Reaction (qPCR) 57
3.2.3. Lentivirus Production 58
3.2.4. Western Blot 58
3.2.5. Cell growth and Viability assay 59
3.2.6. Colony Forming Unit (CFU) Assay 60
3.2.7. Wound Closure Assay 60
3.2.8. Migration Assay 60
3.2.9. Clinical Expression and Survival Dataset 61
3.2.10. Gene Correlation Analysis 61
3.2.11. Enrichr Gene Ontology and Signaling Pathway Analysis 61
3.2.12. Statistical analysis 62
3.3. Results 64
3.3.1. Dutasteride, a 5α-reductase inhibitor, decreases testosterone-AR-induced cell viability and migration 64
3.3.2. Inhibition of 5α-reductase decreases testosterone-associated tumor cell proliferation and migration 68
3.3.3. 5α-reductase inhibition prevents the increase in protein levels associated with tumor progression induced by testosterone treatment 73
3.3.4. Expression and survival rate of steroidal 5α-reductase 1 (SRD5A1) in BCa 76
3.3.5. Silencing of SRD5A1 represses tumor progression in BCa cells 82
3.4. Discussion 85
References 88
Abstract (in Korean) 100
Table 2-1. Bladder cancer driver genes 30
Table 2-2. Nanoparticle Tracking Analysis of urinary exosomes 33
Table 2-3. DNA concentration and absorbance mean values of urinary cell pellet samples from patients with urinary bladder cancer 33
Table 2-4. DNA concentration and absorbance mean values of urine supernatant from patients with urinary bladder cancer 34
Table 2-5. DNA concentration and absorbance mean values of urinary exosomes from patients with urinary bladder cancer 34
Table 2-6. Clinical features of 12 patients with BCa and the method used 35
Table 2-7. Contingency table of number of overlapping variants between patient samples with 12 bladder tumor DNA and paired urine cell pellet DNA 44
Table 3-1. Oligonucleotides used for RT-PCR 63
Table 3-2. Oligonucleotides used for RT-qPCR 63
Figure 2-1. Nanoparticle tracking analysis of urinary exosomes and comparison of urinary DNA samples from patients with bladder cancer. 32
Figure 2-2. Schematic workflow of NGS by superficial bladder cancer tissue and their urinary cell pellet. 38
Figure 2-3. Multidimensional genomic mutational landscape of bladder cancer and cell pellets with coding and non-coding regions. 39
Figure 2-4. Number of overlapping variants in 12 BCa tissues and paired urine cell pellet DNA. 41
Figure 2-5. Mutation hotspots of a single gene from tissues and urinary cell pellets of BCa. 43
Figure 2-6. Somatic alterations in signaling pathways in BCa tissues and paired urine cell pellet DNA. 47
Figure 2-7. Sanger sequencing validation of variants identified using whole-exome and whole-genome sequencing. 49
Figure 2-8. Tumor mutational burden (TMB) analysis between tissue and cpDNA pairs included for comparison. 51
Figure 3-1. Androgen receptor expression and overexpression in bladder cancer cell lines 66
Figure 3-2. Effect of dutasteride treatment on normal bladder cell line and bladder cancer cell lines 67
Figure 3-3. Dutasteride, a 5α-reductase inhibitor, reverses the increase in growth and proliferation of bladder cancer cell lines induced by... 70
Figure 3-4. 5α-reductase inhibition prevents wound closure and migration of bladder cancer cells induced by testosterone treatment 72
Figure 3-5. 5α-reductase inhibition decreases the translational level of cancer progression-related proteins in bladder cancer cells treated with testosterone 75
Figure 3-6. Expression of Steroidal 5α-reductase 1 (SRD5A1) and correlation with survival rate in patients with bladder cancer 79
Figure 3-7. SRD5A2 expression and patient survival plot in patients with bladder cancer 80
Figure 3-8. Signaling pathways of SRD5A1 related with cancer proliferation in urothelial carcinoma 81
Figure 3-9. Effects of SRD5A1 silencing in T24 and J82 bladder cancer cell lines 84