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Title Page
Contents
ABSTRACT 10
INTRODUCTION 12
MATERIALS & METHODS 15
1. Cell culture 15
2. Three-Dimensional culture 15
3. Immunohistochemistry (IHC) 16
4. Transfection of Small interfering RNA (siRNA) 16
5. Transfection of miRNA precursors & inhibitors 17
6. RNA isolation 17
7. MiRNA expression analysis 17
8. Western blotting analysis 18
9. 5-Aza-2'-deoxycytidine treatment 19
10. Purification of genomicDNA and bisulfite treatmentn 19
11. Methylation analysis (Pyrosequencing & MS-HRM assay) 19
12. Lentiviral microRNA systems 21
13. Immunofluorescence 21
14. Statistical analysis 22
RESULTS 23
1. Epithelial-to-mesenchymal transition (EMT) is associated with cyst development in ADPKD 23
2. miR-192 and miR-194 are downregulated in vivo in kidney tissues of both human ADPKD patients and Pkd1 conditional knockout (cKO) mice 29
3. miR-192/215 and miR-194 influence cyst growth 33
4. Epithelial-to-mesenchymal transition (EMT)-associated genes are differentially expressed by miR-192/215 and miR-194 36
5. Downregulation of miR-192 and miR-194 is related to DNA hypermethylation 39
6. Overexpression of miR-192 and miR-194 influences cyst progression in vivo 47
DISCUSSION 55
REFERENCES 58
ABSTRACT IN KOREAN 60
Table 1. Information of antibodies used western blotting 18
Table 2. Primer sequences for methylation-specific high resolution melting (MS-HRM) assay 42
Figure 1. Cyst development of Pkd1 conditional knockout (cKO) mice at all over the age. 25
Figure 2. The differential protein expression levels of epithelial-to-mesenchymal transition (EMT)-associated genes in P13 of Pkd1... 27
Figure 3. Inhibition of cyst growth by knockdown of epithelial-to-mesenchymal transition (EMT)-associated genes. 28
Figure 4. The expression levels of miR-192 and miR-194 in human kidney tissues. 30
Figure 5. The expression levels of miR-192 and miR-194 in Pkd1 conditional knockout (cKO) mice. 31
Figure 6. The expression levels of miR-192 and miR-194 in collecting duct cells isolated from the kidneys of Pkd1 conditional knockout (cKO) mouse. 32
Figure 7. Accelerated cyst growth by inhibition of miR-192/215 and miR-194. 34
Figure 8. Repression of cyst growth by overexpression of miR-192/215 and miR-194. 35
Figure 9. The differential expression of epithelial-to-mesenchymal transition (EMT)-associated genes by miR-192/215 and miR-194 inhibition. 37
Figure 10. The differential protein expression of epithelial-to-mesenchymal transition (EMT)-associated genes by miR-192/215 and miR-194... 38
Figure 11. Bisulfite pyrosequencing analysis of methylation level of hypermethylated MIR-192-194-2 in demethylating agent, 5-aza-dC,... 41
Figure 12. The normalized melting curves of the methylation-sensitive high resolution melting (MS-HRM) analysis of hypermethylated MIR-192-194-... 43
Figure 13. Mature and primary miR-192 and miR-194 expression levels upon 5-aza-dC treatment in WT9-12 cells. 44
Figure 14. The differential protein expression of epithelial-to-mesenchymal transition (EMT)-associated genes in 5-aza-dC treated WT9-12 cells. 45
Figure 15. Proposed schematic diagram on relation among miR-192/215 and miR-194, methylation, epithelial-to-mesenchymal transition (EMT), and... 46
Figure 16. The expression levels of miR-192 and miR-194 in collecting duct cells isolated from the kidneys of Pkd1 conditional knockout (cKO) mice. 49
Figure 17. The expression levels of miR-192 and miR-194 in lentivirus infected mIMCD cells. 50
Figure 18. Injection of lentiviral-overexpressed miR-192 and miR-194 in Pkd1 conditional knockout (cKO) mouse model. 51
Figure 19. The observation of cyst progression in Pkd1 conditional knockout (cKO) mice. 53
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